An Evaluation of Modulating an Ischemic Environment through Angiogenic and Stem Cell Re-

cruitment Chemokine Supplementation in an Effort to Improve Functional Regeneration in Post-

Acute compartment syndrome.

BY

Neel K. Sharma

A Thesis Submitted to the Graduate Faculty Of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

In partial fulfillment of the Requirements

for the Degree of

MASTER OF SCIENCE

Molecular Medicine and Translational Science

5/2017

Winston-Salem, North Carolina

Approved By:

Dr. Shay Soker - Committee Chair

Dr. Tracy Criswell - Advisor

Dr. Jason Hoth- Committee Member

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Table of Contents

List of Figures. 3 List of Abbreviations 4 Abstract: 5 Introduction: 6 Methods and Materials 16 Results: 20 Results VEGF: 20 Results SDF1a 35 Discussions 54 Conclusions 62 Bibliography 66 CV 72

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List of Figures.

Figure 1: VEGF signaling

Figure 2: VEGF 14 day Muscle Function

Figure 3: VEGF Pico-Sirus Red Fibrosis analysis

Figure 4: VEGF H&E mean muscle fiber&muscle fiber composition

Figure 5: VEGF Pax 7 Immunohistochemistry

Figure 6: VEGF Myogenic PCR

Figure 7: VEGF Trichrome blood vessel number and size

Figure 8: VEGF vascular profile PCR

Figure 9: SDF1a 7 and 14 day muscle function

Figure 10: SDf1a 7 and 14 day Pico-Sirus Red Fibrosis Analysis

Figure 11-13: SDf1a 7 and 14 day inflammatory PCR.

Figure 14: SDF1a 7 and 14 day H&E mean muscle fiber Stable and regenerating

Figure 15: SDF1a 7 and 14 day H&E muscle fiber composition

Figure 16: SDF1a 7 and 14 day Pax 7 Immuno-histochemistry

Figure 17: SDF1a 7 and 14 day myogenic PCR

Figure 18: SDF1a 7 and 14 day Trichrome blood vessel number and size

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List of AbbreviationsCS-CSVEGF- Vascular endothelial growth factor A isoform 165SDF1a/CXCL12-Stromal cell derived factor 1 alpha/chemockine ligand 12HEK cellsVPF-vascular permeability factorflt-1-fms-like tyrosine kinase 1, VEGFR1KDR-kinase insert domain, VEGFR2AKT-protein kinase BNP-1-neuropillin-1SH- Src Homology domainSh2 receptor- receptor for SHMIp-1alpha-macrophage inflammatory protein 1 alphaCXC motif-chemokine motifRFFESH motif-protein residues 12-17 in CXCL12CXCR4-chemokine receptor 4HIV-human immunodeficiency virusE-selectin-Endothelial leukocyte adhesion moleculeP-selectin-Platelet Activation-Dependent Granule to External Membrane Protein (PADGEMVCAM-vascular cell adhesion molecule 1ICAM-Intercellular Adhesion Molecule 1VIVA trial-Vascular endothelial growth factor in Ischemia for Vascular AngiogenesisWAG rats-wistar albino galaxo DM-Differentiation mediumflk-1-fetal liver kinase-1DRG-Dorsal Root GanglionSCG-superior cervical ganglion

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Abstract:Compartment syndrome (CS) is a condition in which osseofacial plane pressure

increases in response to local injury enough to block venous outflow and lead to muscle

ischemia, necrosis, and fibrosis. The injury usually involves the upper or lower extremi-

ties and can lead to limb amputation or paralysis. The treatment for CS is fasciotomy

though this treatment is time-limited and comes with significant sequelae. In the time af-

ter fasciotomy is effective, no current treatment modality exists to recover muscle func-

tion. The pathophysiology of CS can be amenable to biologic therapy that falls outside

the therapeutic window of fasciotomy to recover muscle function. We studied the use of

the intramuscular injections of biologic’s VEGF and SDF1a to attempt to reverse the CS

environment and recover muscle function.

Methods: CS was induced according to the protocol set up in Criswell et al [1].

Treatments were administered as intramuscular dosages 100ul (500ng/ul) for VEGF at

day 3,7,and 11 and 50ul (100ng/ul) at the time of injury for SDF. Muscle function, histol-

ogy, and QPCR was performed to evaluate the efficacy of therapy.

Results: As compared to Saline treated injured muscles, VEGF treated injured

muscles showed a significant increase in muscle regeneration between day 7 and day

14, higher mean stable fibers, lower mean degenerative fibers, higher numbers of ves-

sels per high powered field (p<0.05). In SDF1a treated muscles there was a significant

decrease in degenerating fibers at day 14, significantly increased pax7 positive nuclei

per high power field at day 7 and day 14, and significantly decreased percent area fibro-

sis at day 7 and day 14 when compared to saline (p<0.05).

Discussion: VEGF and SDF1a provide novel ways to increase regeneration pa-

rameters in the compartment model though neither was able to adequately affect muscle

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function. More studies are needed in terms of delivery mechanisms, timing of delivery,

and synergistic effects of biologics to increase therapeutic efficacy in the CS model.

Introduction:

Compartment Syndrome (CS) was first observed 130 years ago in Volkman’s re-

search into lower extremity injury resulting in paralysis and amputation [2]. 130 years

since CS was characterized there has been further pathophysiologic information in the

disease process but practical management strategies have remained difficult [2] [3] [4] .

The pathophysiology of CS is defined by limb injury mediated increase in osseofacial

plane pressure leading to decreased venous outflow, hypoperfusion, muscle ischemia,

accumulation of reactive oxygen species, cell death and finally fibrosis [4]. The current

standard of care is fasciotomy [5],[6],[4] which involves surgically increasing the osseo-

facial plane space to decrease intra-compartmental pressure. This procedure is time limit-

ed and caries significant adverse sequelae such as extensive scarring and long term mus-

cle weakness [3],[4].

Because the damage from CS is caused by hypoperfusion and results in ischemic

fibrosis, the need for therapies which can increase perfusion, angiogenesis, and provide

regenerative cells necessary to recover from injury is integral. The two agents we utilized

to understand the therapeutic effect of biologic modulation of the CS environment are

Vascular Endothelial growth factor A isoform 165 (VEGF), and Stromal-cell derived fac-

tor 1 alpha (SDF1a/CXCL12).

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VEGF was used in this study due to it’s ability to increase angiogenesis and en-

dothelial survival. In the CS model, ischemic injury is the cornerstone of cell damage and

could be mitigated through increasing the vascular regenerative abilities of the native

tissue. The structure and function of VEGF is integral to understanding it’s therapeutic

utility. VEGF is a dimeric growth factor that was first studied in embryogenic and tu-

morigenic models. VEGF was first discovered in 1980 [7], and is included in a family of

VEGFA, B, C, D, E, and F as well Placental growth factor [7], [8], [9] , [10], [11]. VEGF

A is the most studied form of VEGF. VEGFA is additionally divided into isoforms which

are formed through differential splicing of transcripts. The sizes of these transcripts de-

termine the nomenclature of the VEGF isoforms and include VEGF 189, 165 and 121

[12]. VEGFA 165 is considered the most physiologically active and the most pro-angio-

genic [13], [11].

The function of VEGF was first discovered through in vivo studies involving the

transfection of HEK cells with a VEGF vector [14]. After the vector was transferred to

HEK cells, the supernatants of cells were collected [14]. When this supernatant was incu-

bated with capillary endothelial cells, vascular sprouting resulted. However, along with

the sprouting of capillary endothelial cells [14], it was also found in further study that

VEGF caused extravasation of evans blue dye from treated guinea pigs[15]. This led to

the alternative naming of VEGF as vascular permeability factor or VPF [15].

VEGF action in cells is mediated through tyrosine kinase mechanisms. The cell

surface receptors for VEGF, VEGFR1-3, are the entry way into the angiogenic effects.

VEGF receptor 1 (VEGFR1) also known as flt-1 is a membrane bound tyronsine kinase

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receptor [16], [13]. This receptor has the highest affinity for VEGF and the lowest tyro-

sine kinase action upon binding [16]. The proposed purpose of this juxtaposition is to of-

fer a temporizing effect to circulating VEGF [16]. VEGFR2 also known as kinase insert

domain receptor (KDR) is also a membrane bound tyrosine kinase receptor and has the

highest angiogenic potential upon VEGF binding [13], [17], [18]. The regulation of this

action involves constitutive endocytosis and degradation of VEGFA and VEGFR2 pro-

teins upon ligand-receptor binding [19]. VEGFR3 is mainly expressed in embryonic

lymphatic vessels and is involved in lymphogenesis, and ,interestingly, is not actively in-

volved in vessels that are in an injured environment [20]. VEGF can also bind to a cell

surface glycoprotein known as neuropillin-1 [21]. NP-1 has the ability to activate AKT

when bound to VEGF [21]. Soluble NP-1 has been shown to have anti-tumor activities on

tumorigenic VEGF [22].

VEGF signal transduction occurs through VEGFR binding and dimerization in a

tyrosine kinase activation cascade. This leads to phosphorylation of Src homology 2 SH2

receptor domains or phosphotyrosine binding domains (PTB) of the VEGFR [23]. Phos-

phorylation of the PTB domain leads to further signal transduction and,in short, leads to

increased endothelial proliferation, migration, and survival [23]. The outline of this

process is listed in figure 1 [13].

Stroma-Cell derived factor 1 alpha (SDF1a/CXCL12) is another biologic which

can have potential utility in the CS model and represents an alternative way to increase

the regenerative capabilities of injured muscle. SDF1a functions as a chemokine, or a

protein which can recruit cells to an area in which it’s concentration is increased. The

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uniqueness about SDF1a, and the heart of it’s therapeutic utility in the CS model, lies in

the ability to recruit stem cells which contain it’s receptor. When stem cells are aggregat-

ed in an area of injury, in our case CS injury, the potential for regeneration is increased

[24]. Again, further understanding of the structure and function of SDF1a is integral in

understanding it’s therapeutic utility in the CS model.

As a member of the chemokine family SDF-1a has shared structural chemokine

characteristics and a few key differences. The similarities between SDF1a and other

chemokines in this family include a tertiary fold with an N-terminal region, a CXC motif

loop region, three antiparallel B strands and a C-terminal alpha helix [25]. SDF is differ-

ent in its hydrophobic core with Trp57 oriented towards the first B-strand, similar to in-

terleukin-8 [25]. The N-terminal region and RFFESH motif on residues 12-17, deter-

mines the specificity and response potency of the SDF-1a binding to it’s receptor CX-

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Fig1

Cmotif receptor 4 (CXCR4). This was discovered through experimentation involving

SDF1a with an N terminal region missing only a terminal lysine. When this modified

SDF1a molecule was studied in CD34+ cells, signaling through its receptor, CXCR4,

was unable to proceed [25] [26] [27]]. SDF1a molecules with an intact RFFESH region

in the N terminal region had no issue with signal transduction through CXCR4 [25]

The primary receptor for SDF1a, CXCmotif Receptor 4 (CXCR4), is part of the

G-protein coupled receptor super family [28] . G-protein coupling was elucidated in an

experiment involving tissue exposed to pertussis toxin resulting in the cessation of SDF1a

signaling[29]. Although a member of the chemokine receptor family, CXCR4 lacks the

heterogenous receptor selectivity of other members of the family. This fact is problematic

for therapeutic utilization but also becomes catastrophic when known that is is a major

binding and entry site for Human immunodeficiency virus 1 (HIV-1)[28] . The known

activators of CXCR 4 are SDF1a, chimeras of SDF1a, and HIV adhesion molecule gp120

[28]. Inhibition of CXCR 4 is accomplished through a peptide antagonist (CTCE-9908),

and a nonpeptide antagonist (AMD3100) [30]

Signal transduction through the CXCR4 pathway is less understood than VEGF

signaling through the VEGFR. Studies have shown that when exposed to optimal concen-

trations of SDF1a in vitro (100-300ng/mL), CD34+ cells migrate to where SDF1a con-

centrations are the highest[28, 31, 32]. The two theories of migration are centered around

either PI3k/Akt or MAPK/ERK signaling in cells that bind SDF1a. Studies have shown

that when CD34+ cells were pre-treated with PI3k inhibitor LY294002 that no migration

was able to be induced in CD34 cells at optimal concentrations of SDF1a. MEK inhibitor

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PD98059 did not show this effect [33]. Further downstream signaling is dependent on

Akt phosphorylation of eNOS and NO generation. When eNOS was inhibited with N-ni-

gro-arginene methyl ester, (L-NAME) there was significant attenuation of CD34+ migra-

tion. This migration is seemingly regulated through the PI3K/Akt pathway though there

are studies which have shown that MAPK/ERK signaling is involved in CXCR4 up-regu-

lation indicating some role on chemotaxis [31]. Studies to greater determine the effect

transduced by SDF1a binding to CXCR4 are ongoing [32] [34], [31].

Originally thought to be limited to the bone marrow stromal cells [35], SDF1a

has since been found to be secreted by endothelium, and platelets [36]. Signal transduc-

tion studies involving SDF1a binding to CXCR4 have allowed a greater understanding of

SDF1a function. It was found that when exposed to optimal concentrations of SDF1a,

between 100-300ng/mL, hematopoetic stem cells (CD34+/CXCR4+ cells) were found in

greater concentrations in vitro as compared CD34+ cells exposed to another known stro-

mal cell secretion, MIP-1alpha in vitro [33], [29].

Another action of SDF1a that facilitates honing of CD34+ cells to injury, is the

ability to increase the intravascular adhesive properties of these cells when exposed to

SDF1a. For a CD34+ cell to exert action it must be able to leave the vasculature and en-

ter the site of injury [37]. Adhesion molecules which have traditionally been considered

in the vasculature include E-selectin (Endothelial leukocyte adhesion molecule) [38], P

selectin (Platelet Activation-Dependent Granule to External Membrane Protein

(PADGEM))[39], and, in the absence of selectins, VCAM and ICAM [40]. These are ex-

pressed intravascularly to allow cells to extravasate. A study by Pedel et al has shown

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CD34+ cells adhere to integrin ,leading to cellular locomotion arrest, to a greater degree

when SDF1a exerts its action on the ICAM receptor [37] [41].

Characterization of VEGF expression during ischemic injury became an important

first step in understanding how to utilize it’s effectiveness. Takashita et al looked at is-

chemic injury in Rabbits and noted that VEGF and VEGFR2 (KDR) expression was in-

creased in those rabbits suffering acute ischemia. When chronic ischemia was studied,

however, KDR expression was only noted in those cells which were actively regenerat-

ing. This was hypothesized to be occurring through a paracrine effect of muscles in regu-

lating the exposure to VEGF [42] . Van Weel et al studied the expression of VEGF in pa-

tients with chronic limb ischemia and found that during exacerbations or “acute-on-

chronic” ischemic events the expression of VEGF in vascular structures and myofibers

increased threefold [43]. When studying the native ischemic environment, non-acute,

VEGF levels were decreased or unchanged. These findings indicated that the native re-

sponse to new onset ischemia is to upregulate VEGF to restore perfusion. The implication

here is that modulation of the native response in ways that enhance the innate vascular

recovery mechanism is a possible treatment modality for ischemic diseases such as com-

partment syndrome [44], [45], [46], [42], [43].

When these expression profiles were discovered, animal models of ischemia and

treatment with VEGF was conducted. Myocardial infarction studies had shown success-

ful co-lateralization of coronary arteries with VEGF administration to ischemic tissue

[47], [48], [49], [50]. The Phase 1 VIVA ((Vascular endothelial growth factor in Ischemia

for Vascular Angiogenesis) trial which studied 178 patients with stable non-exertional

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angina who were not revascularization candidates, showed significant improvement in

Angina class in the high dose recombinant VEGF165 intracoronary infusion group at 120

days [51].

Limb ischemic studies have shown improvement at 28 and 30 days post injury

when VEGF was injected intravenously or intramuscularly[52]. Specifically in rabbits, it

was noted that, in an arterial ligated hindlimb ischemia model, the high dose VEGF

treatment group (1000mcg+heparin), led to significant capillary density increase and sig-

nificantly higher angiographic score at day 30 [52]. Also noted, when VEGF was admin-

istered in 10 day daily intramuscular injections to an ischemic environment, clinical im-

provement in the degree of muscle atrophy and necrosis was lower at day 30 in the high

dose VEGF (1000mcg) group. ([53]). Walder et al also noted that in an arterial ligation

model of ischemia, after a single IV injection of VEGF, muscle blood flow was signifi-

cantly improved when compared with vehicle treated controls at 28 days [54].

Like VEGF, SDF1a has had similar promising functional results when looking at

it’s therapeutic utilization in ischemic injury. Brzoska et al evaluated SDF1a use in

hindlimb crush injury involving WAG Rats [55]. It was found that, when 100ng/ul SDF1a

was administered in two 10ul injections at the time of injury, there was significant in-

crease in muscle mass, decrease in fibrosis, and better tissue architecture noted on histol-

ogy at day 7 and day 14. Increases in the expression profile of healthy muscle activity

markers, myosin heavy chain and muscle creatinine kinase, were also noted [55] Tan et al

studied a vector delivery model of SDF1a therapy in ischemic injury. Five treatment

groups were included in this model and were as follows: Group 1 was injected (I.M.)

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with 0.1 ml of saline (0.9% NaCl). Group 2 was injected intraperitoneally (I.P.) with G-

CSF. Group 3 was injected I.M. with NIH 3T3 engineered to express LacZ (NIH 3T3/

LacZ) plus injection I.P. of G-CSF. Group 4 was injected I.M. with NIH 3T3/SDF-1.

Group 5 mice were injected I.M. with NIH 3T3/SDF-1 plus injection I.P. of G-CSF.

Combination SDF NIH3T3 and GCS caused the greatest increase in perfusion at 3 weeks

via laser doppler analysis, compared with either treatment alone, or with saline alone[56].

These results have also been noted in other studies involving SDF1a in ischemic injury

[57], [58], [59], [60].

The structure and function of VEGF and SDF1a have been elucidated using in

vitro experiments, and therapeutic utility has been noted in vivo experiments, however

beyond the well noted primary effects of these biologics, other influences on cells have

been delineated. The CS injury that we induce leads to a multifactorial hindlimb injury

including disruptions to musculature, vasculature, and nervous architecture. It is thereby

important to understand as many of the secondary effects of our biologic interventions as

possible.

VEGF has long been studied in terms of it’s action on endothelial cells, though

recent evidence suggests that it’s influence can be found in other non-endothelial cell

populations [61], [62], [63], [64], [65], [66]. VEGF influence on Skeletal muscle progen-

itor cells (satellite cells) is particularly intriguing owing to the presence of VEGFR1&2

on these progenitor cells [64]. Germani et al showed that when myogenic progenitor

cells were cultured in differentiation medium (DM), the apoptotic rate at 3 days was

10.6% of total cells cultured. When VEGF was supplemented at 20ng/ml the percentage

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of apoptotic cells significantly decreased from 10.6% to 7% [67]. These results mir-

rored other studies into the survival effect of VEGF on muscle progenitor cells [68], [69],

[70]. SDF1a also has been studied in terms of its muscle regenerative capabilities though

much less research has been conducted on its effects. For example, Melchionna et al

showed that when CXCR4 was blocked myogenic differentiation was unable to proceed

[71].

Our injury model goes beyond simple ligation of artery or toxin injection to repli-

cate the ischemic pathophysiology of CS. The compressive model put forth by Criswell et

al 2012 has been used to replicate the dynamic injury environment through replicating the

initial insult of decreased venous outflow and increased of CS going beyond the tradi-

tional ligation or toxin methods[1]. Our animal selection for these experiments were

Male Adult Retired Breeder Lewis Rats, We picked this model in these rats as, in lower

order animals, muscle damage was quickly recovered from (mice exhibited increased re-

generative potential, and smaller muscles) allowing no viable therapeutic intervention

window. Young Rats exhibited similar regenerative capabilities to mice and were ruled

out as well. Adult rats aged 10-12 months provided a wide therapeutic window, analo-

gous CS injury environment, and allowed for ease of injury induction [1]. The knowledge

gained from the ligation and toxin methods however showed the utility of biologics in

these ischemic environments. Flowing from these studies we propose that after the induc-

tion of CS, beyond the efficacious time point for fasciotomy, restoration of perfusion, in-

creasing stem cell material, and promoting muscle cell survival and differentiation will

lead to a recovery of muscle function.

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• My research question is ‘In a CS environment, will the proposed mechanisms of

VEGF and SDF1a action en-vivo lead to attenuation or possibly reversal of the multi-

factorial CS injury to ultimately retain muscle function?’

• Will VEGF or SDF supplementation maintain the integrity of muscle architecture

in the CS environment?

• Is bolus delivery of biologics a viable method to treat CS?

• Will supplementation of angiogenic factors VEGF or increasing the stem cell ma-

terial through SDF1a injection cause a change in muscle function?

Methods and Materials Animals

Animal studies were performed according to protocols and guidelines set forth by the

Wake Forest University Animal Care and Use Committee and NIH standards. Le Male

Lewis Rats aged 10-12 months old were used in these studies (Envigo, Cambridgeshire,

UK). Animals are individually housed in climate controlled environments and are given a

week to acclimate before any interventions are performed.

CS Induction

CS induction will be accomplished according to protocol set up by Criswell et al

which had put forth the protocol for and similarities between CS pathophysiology in hu-

man and the model injury characteristics [1]. Prior to induction, Lewis rats aged to ap-

proximately 12 months were anesthetized through inhaled Isoflourane in an anesthesia

chamber as well as maintained in nose cone overtop a heated water pad. After anesthesia

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is confirmed, the left hindlimb of each rat is shaved and weight based dosing of ketofen is

administered intraperitoneally to prevent post operative pain. After the left hindlimb is

prepped, neonatal blood pressure cuffs are placed on the left hindlimb proximal to the

location of the anterior compartment. . Cuffs placed on the left hindlimb are then inflated

to pressures which are manually maintained at 120-140 mmhg. These pressures are con-

tinued for 3 hours to mimic the increased venous outflow pressure pathopneumonic for

CS. During CS induction 1cc of physiologic saline is administered intraperitoneally at

one hour intervals to maintain hydration. After 3 hours of perfusion cessation is complet-

ed rats are removed from isofluorane and observed until the effects of anesthesia have

subsided. Rats are then returned to cages and monitored daily for three days to determine

if additional pain control is needed.

Biologic preparation

VEGF preparation is performed through reconstituting powdered VEGF in phys-

iologic saline to a concentration of 500ng/ul. Heparin is added to the VEGF solution to

increase bioavailability and prevent degradation. VEGF injection volumes were 100ul,

and delivered intramuscularly to the ischemic hindlimb. SDF1a is reconstituted through

the same procedure to a concentration of 100ng/ul though heparin is not included. Injec-

tion volume is 50ul for SDF1a.

Injections are performed at day 4,7,and11 for VEGF. In the SDF1a experiments 1

injection at the time of injury is performed. This injection is delivered to the ischemic

hindlimb immediately after CS induction.

Muscle Function

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I assessed muscle function through force of dorsiflexion in the left hindlimb at

frequencies designed to test muscle twitch-tetany (1hz-200hz). In short, first the Lewis is

weighed to determine the baseline and to measure if the rat is acutely distressed by com-

paring to weights at day 7 and day 14. Rats are then prepped for the procedure through

Isofluorane anesthesia induction and maintenance through nose cone inhalation. After

anesthesia is performed the left hindlimb is shaved and prepped with three alternating

swabs of iodine and isopropyl alcohol. Eye drops are placed to increase comfort in the

animal. After the animal is prepped it is placed in the muscle function apparatus. The

limb is positioned at a right angle to the base of the apparatus and held in place in the

force recording foot pedal. Electroprobes are placed in the region of the anterior com-

partment which will stimulate the tibalis anterior and the extensor digitorum longus. This

action is achieved through stimulating the peroneal nerve which innervate the two major

dorsiflexors of the lower limb. The voltage is set between 1.8-2.2 mV to achieve and ide-

al contraction curve. The maximal force is then measured through the LabView-based

software program (provided by the US Army Institute of Surgical Research), and record-

ed for analysis. After analysis the rat is observed for recovery from anesthesia and placed

in it’s climate controlled cage. Muscle function is performed at baseline (7 days before

CS induction), at day 7 and day 14 after CS where applicable.

Histologic Preparation

At the terminal days (day 7 or day 14) of study rats are sacrificed and tissue col-

lected. Muscle tissue is collected from the Tibialis anterior and extensor digitorum longus

of both the injured and uninjured limb. The tissue is weighed and placed in alcohol for

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fixation. After tissue is fixed for 1 week after which it is parafin embedded and sectioned

into 8um slices. These slices are then placed on slides for drying, staining and microscop-

ic evaluation. Various staining is used to capture different aspects of the muscle during

regeneration. Hematoxylin and Eosin staining is performed to evaluate muscle cell mor-

phology, architecture and inflammatory infiltration. Mason’s Trichrome staining is per-

formed to evaluate for vessel number and size. Sirus Red staining is used as well to eval-

uate for fibrosis through collagen percentage. Immunohistochemistry is performed to

evaluate for satellite cell pax7+ nuclei per high powered field and is used as an analog for

regenerating muscle tissue.

Quantitative PCR

RNA was isolated from muscle tissue using The PerfectPure RNA Fibrosis kit as well as

the Quiagen Fibrous RNA isolation kit according to the manufacturer’s protcol. cNA was

prepared using the High Capacity cDNA Reverse transcription kit (Applied Biosystems,

Foster City, CA). Quantitative PCR was performed in 20-ul Reactions using 96 well

plates using 10ul of RNA and PCR safe water, concentrations of each determined by the

purity of the RNA isolated per sample. 10uL included master mix, taqman probe and ran-

dom primers, to a total of 20ul per well. Taqman probes for housekeeping gene GAPDH

(Rn01775763_g1), Pax7 Rn01518732_m1, MyoD, MyoG, CD31 (VEGF), KDR

(VEGF), TGF-B1 Rn01475963_m1 (SDF1a), Col1a1(SDF1a) were used.

Statistics

Functional analysis was performed using a two-way ANOVA, morphological studies

comparing uninjured muscle, control treatment, and interventional treatment were per-

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formed using Two Sample two tailed independent T-tests to determine significancy. All

statistical analysis is compared to a p value of 0.05 to achieve significance. Statistical

analysis was performed using native programs in Numbers/Excel (Redmond, WA). and

Graph Pad Prism Software (GraphPad Software, San Diego, CA)

Results: VEGF and SDF1a were used in the CS model due to regenerative recovery data

demonstrated in previous hindlimb ischemic models. CS is different from simple is-

chemic models due to the multifactorial arterial, nervous, and muscle involvement. To

test if the promising recovery from ischemia data translated to recovery in the CS model

we administered intramuscular biologic intervention either at the time of injury (SDF1a),

or at selected intervals after injury (VEGF).

Results VEGF: Muscle Function

Muscle function was analyzed at baseline 7 days prior to injury and day 7 and

day 14 after injury. Electro-probe peroneal nerve induced force of dorsiflexion was mea-

sured and converted to isometric torque using weight and force conversion. At day 7 av-

erage saline muscle function was greater at all frequencies (twitch 1hz to tetany 200hz)

though this result was not statistically significant. The same held true for day 14 which

again was not statistically significant. However when day 7 to day 14 relative growth was

analyzed muscle function was increased when compared to saline, at 10hz this result was

statistically significant (p=0.0187).

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Histologic Examination: Pico-Sirus Red

In the CS model fibrosis is a sign of end organ damage and irreversible muscle

loss. Preventing fibrosis is an important way to mitigate damage of CS. We evaluated fi-

brosis through Pico-Sirus red staining for collagen on 8um slides of which a minimum of

5 images per animal muscle were evaluated. Uninjured, Saline injured and VEGF injury

muscle tissue were used in this analysis. Results are depicted in figure 3, but It was found

that there was no significant difference in percent area fibrosis when comparing VEGF

treated muscle tissue and Saline treated muscle tissue at 14 days (7.2 vs 6.95 p=0.85).

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Fig 3. Pico-Sirus Red Fibrosis Analysis. Mean percentage area fibrosis analyzed through Image J measurements.

H+E

Muscle samples were collected, processed and parafin embeded according to pro-

tocol. 8um sections of muscle tissue were obtained and imaged after staining with hema-

toxylin and eosin per protocol. At least 5 Images were gathered from these slides random-

ly. At least 5 images per animal muscle were analyzed. Results are displayed in Fig 4, but

when comparing VEGF and Saline treated injured muscle tissue, VEGF treated animals

had a significantly higher number of mean stable fibers (147 vs 129 p=0.029), and de-

creased degenerating fibers (10 vs 28 p=0.011). Regenerative fibers, described as fibers

with central nuclei by convention, were increased in VEGF treated animals when com-

pared to saline but this result was not statistically significant (35 vs 26 p=0.32). The

composition percentage analysis shows a healthier muscle tissue based on increased per-

centage of stable and regenerative fibers with a decreased percentage of degenerative

fibers.

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Fig 4. H+E examination of injured muscle tissue. A. Mean number of fibers. Muscle fibers delineated by the following criterion. Central nuclei > 3 Degenerative, Central Nuclei <3 Regenerative, No central nuclei Stable. Significance **p=0.01, * p=0.05. B. Percent composition of Muscle Fibers, total compared to individual categories of muscle fibers.

Immunohistochemistry: Pax7 Analysis

Satellite cells are muscle progenitor cells which are involved in muscle differenti-

ation and recovery from injury. These stem cells are able to undergo myogenic differenti-

ation and thus are a useful analog to the extent of muscle recovery. These cells are typi-

cally positive for myogenic marker Pax7. Pax7 immunohistochemistry was performed to

quantify populations of these cells in the injured tissue at the 14 day tissue collection

point. 8um samples were sectioned and stained using immunohistochemistry protocols.

At least 5 images were analyzed per muscle tissue obtained. Pax7 positivity was quanti-

fied using Image J cell counter counting Pax7+ nuclei per high powered field. Results are

depicted in figure 5 but it was found that although there was an increase in mean number

of pax7+ nuclei in the VEGF treatment group this was not found to be statistically signif-

icant (17 vs 15 p=0.32).

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Fig 5. Pax7 Immunohistochemistry. Mean number of nuclei counted through ImageJ processing. ** significant to p=0.01

Molecular Analysis: Myogenic Differentiation QPCR

As mentioned earlier Pax7 is a marker for satellite cell involvement and progeni-

tor stem cell concentration, but muscle regeneration is a complex process with multiple

intermediaries. Among these intermediaries are MyoD a protein involved in early my-

otubule formation, and MyoG (myogenin) a protein involved in late myotubule formation

and integration into a functional unit. We evaluated transcripts from these muscle markers

to evaluate how treatment with VEGF modulated the expression profile of the intermedi-

aries. We found (fig 6) the relative expression of muscle regenerative markers as com-

pared to GAPDH to be increased but not significantly in VEGF treated muscle as com-

pared to Saline (Pax7 p=0.24, MyoD p=0.24, MyoG p =0.15).

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Fig 6. Myogenic differentiation QPCR Analysis. A. Pax7 QPCR analysis as %GAPDH. B. MyoD QPCR as %GAPDH. C. Myo G expression as %GAPDH. Note scale

Trichrome

Trichrome staining was performed to evaluate blood vessel size and number on

8um sectioned slides. At least 5 muscle images per animal were taken and analyzed

through ImageJ cell counter and measuring tools. Vessel size was measured from great-

est diameter across the tunica adventitia. Typically uninjured muscle has smaller numbers

of blood vessels per high powered field, and variable sizes of blood vessels comprised of

either very large arterioles, or small capillaries. Results are summarized in figure 7 but in

short, in VEGF treated muscle there was a significantly higher number of blood vessels

per high powered field , as compared to saline (3 vs 1 p=0.001). In terms of vessel size,

there was no significant difference between VEGF and saline treated muscles (73um vs

75um p=0.80).

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Fig 7. Trichrome blood vessel analysis. All images scaled from 1600x1200 pixels using ImageJ measurements. A. Blood vessel number counted significance of **p=0.01. B.

Molecular analysis: Vasculature

Given that there were a higher number of vessels in VEGF treated injured muscle

tissue we decided to evaluate the molecular profile of the vasculature. Endothelial marker

CD31 and VEGFR2 KDR was evaluated through qPCR. We found that endothelial cells

were higher in the VEGF treated group per trichrome staining and Image J quantification.

Endothelial cell marker expression however was increased in VEGF treated animals

though this result was not found to be significant when compared to the saline treated

group (p=0.11). KDR was found to be similarly expressed in VEGF treated muscles and

Saline treated muscles (p=0.85) (fig 8).

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Fig 8. Endothelial QPCR. A CD31 QPCR as %GAPDH. B. VEGFR2 (KDR) as %GAPDH. Results not significant unless otherwise

Results SDF1a Animal Safety:

During the course of the 14 day SDF1a experiment one animal succumbed to injury and

was euthanized after greater than 10% bodyweight was lost. The animal was euthanized

according to protocol and autopsy was performed to determine cause of death. There

were no signs of malignancy or any other overt pathology to attribute the rapid weight

loss to other than the CS injury. Otherwise all animals tolerated injury and treatment with

minimal additional pain control necessary. Muscle function results are from the same co-

hort of animals, the 14 day injury group. Histology are obtained from two different co-

horts, one followed over 7 days, and one followed over 14 days.

Muscle Function Muscle function was assessed at baseline 7 days prior to injury, and at days 7 and

14 after injury. Maximal force of dorsiflexion elicited by electroprobe stimulation of the

peroneal nerve was measured in units of Nm and converted into torque using animal

weight data. Baseline muscle function was collected and used as the basis to determine

muscle function regeneration by converting day 7 and day 14 muscle function into

%baseline. The results are depicted in figure 10 but in summary, Day 7 muscle function

in terms of mean %baseline was lower in SDF1a at the lowest frequencies (twitch) but

showed higher, average muscle function as more muscle fibers were recruited at higher

frequencies (tetany). These results were not significant. At day 14 muscle function was

lower for all frequencies in SDF1a treated muscle as compared to saline treated muscle.

This result was also not significant. When comparing growth between day 7 and day 14

�35

between treatment groups, Saline again had a higher degree of muscle function when

compared to SDF1a treated animals.

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Fig.9 Muscle function analysis. A. Day 7 Muscle Function. B. Day 14 Muscle Function. C. Day 14/Day7 used to assess

the regeneration between these time points as compared

to %Baseline.

C

BA

Histologic Analysis: Fibrosis analysis

Fibrosis is produced by fibroblast infiltration into the injured muscle tissue. Early

on fibrosis is useful in providing a scaffold to align muscle fibers, but late in disease can

turn into scar formation and is usually a sign of end stage damage in organs and tissue

[72]. It replaces functional tissue with typically permanent scar tissue which acts as a

hindrance to muscle recovery. The CS model has fibrotic replacement of muscle tissue as

an endpoint. To determine fibrosis in treated animals I used Pico-Sirus red staining on

8um sectioned treatment group muscles, and uninjured controls. This stain is specific for

collagen involved in fibrotic lesions (type 1). At least 5 images per animal were ana-

lyzed, and fibrosis quantified using Image J software. It was found that at 7 days the

SDF1a injury group had a significantly decreased mean area of fibrosis when compared

to the saline injury group (p < 0.01). The 14 day cohort experiment showed SDF1a treat-

ed muscle tissue with a significantly lower %area fibrosis as compared to saline treated

muscle tissue (p < 0.01) (fig 10).

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Fig 10. Pico-Sirus Red Fibrosis Analysis. Mean percentage area fibrosis analyzed through Image J measurements.

Molecular Analysis: 7 and 14 day Inflammatory QPC

Given the decreased Fibrosis noted on Pico-Sirus red imaging and quantification,

further analysis of the fibrotic environment was warranted. I studied transcripts for TGF-

B, Col1a1, CD68 and CD163 as these have varying functions in the fibrosis environment.

TGF-B is secreted by tissues in response to injury either through stretch, shear loading, or

ischemia. The result of its secretion is increased matrix protein synthesis and lessened

matrix protein degradation. The end result is fibrosis[73]. Results are depicted in figures

11-13 but in short, in terms of TGF-B, the 7 day study showed a higher level of TGFB

expression in Saline treated animals as compared to SDF1a treated animals though this

was not found to be statistically significant (p=0.42). Day 14 studies showed a slight non-

significant increase in TGF-B expression in SDF1a treated animals as compared to saline

treated (p=0.80). Collagen deposition is the fibrotic material in the CS model. In our par-

ticular model collagen 1 alpha 1 (col1a1) is used to quantify the expression of this fibrot-

ic collagen. At day 7 it was found that in SDF1a treated animals there were less col1a1

transcipts as compared to saline treated muscles though this was not significant (p=0.41).

At day 14 this trend was flipped with SDF1a treated animals expressing more type 1 col-

lagen transcripts than saline, again this result was not significant (p=0.26).

Macrophages are also important in the ischemic environment and infiltrations are

numerous in histology of the injured CS muscles[1]. Macrophage markers also determine

the stage of inflammation the injured tissue is at, with CD 68 a general sign of

macrophage involvement and CD 163 is a sign of anti-inflammatory, typically late,

macrophage involvement. CD 163 is has been shown to be able to internalize TNF-super-

�40

family molecules[74], and is thought of as anti-inflammatory macrophage [75]. In our

injury model, At day 7 and day 14 there was greater average expression of CD68 in SD-

F1a treated animals as compared to Saline treatment groups though this result was not

significant(Day 7 p=0.58; Day 14 p=0.35). At day 14 the average expression of CD163

was higher in SDF1a treated animals though these results were not significant (p=0.34).

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Fig 11. Inflammatory expression QPCR. A. Day 7 TGFB, and Cola1a. B Day 14 TGF-B and Cola1a

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Fig 12. Inflammatory expression QPCR. Non-specific macrophage marker CD 68 and day 7 and

Fig 13. Inflammatory expression QPCR. A. Late stage anti-inflammatory macrophage marker

For H&E analysis, muscle samples were collected, processed and parafin embed

according to protocol. 8um sections were cut and imaged after staining with hematoxylin

and eosin according to protocl. At least 5 random images of muscle tissue from all injured

rats were gathered and analyzed using ImageJ. Results are displayed in Figure 14-16.

H&E data comparing controls to injured muscles showed interesting results. At

day 7 when comparing mean stable fibers of the Uninjured muscles (R hindlimb) of the

various treatment groups (Saline and SDf1a), it was found that there was a significant

increase in mean stable fibers in the Saline Uninjured muscle compared to uninjured SD-

F1a muscles (161 vs 191 p=0.01). For this reason, treatment groups were compared to

their respective uninjured muscles, rather than averaging muscle fibers from all uninjured

muscles (Saline R Hindlimb vs Saline L hindlimb, SDF1a R hindlimb vs SDF1a L

hindlimb). In saline treated injured muscles there was a significant decrease in mean sta-

ble fibers compared to saline treatment group uninjured muscles (130 vs 191 p=0.0001)

at day 7. In SDF1a treated animals there was no significant difference in mean stable

fibers between SDF1a uninjured and injured muscles (150 vs 161 p=0.39) at day 7 show-

ing an attenuation of injury. This trend in differing uninjured mean muscle fiber composi-

tion between treatment groups did not hold true when comparing mean degenerating and

regenerating fibers and thus uninjured mean composition of these fibers were averaged

together and compared to treatment groups. Regenerating fibers were both predictably

increased when compared to uninjured animals for both treated injured muscle groups

(SDF1a p= 0.03, Saline p=0.01), though surprisingly there was no significant difference

in degenerating fibers when treated injury muscle were compared to uninjured muscle

�44

Histologic Analysis: H+E 7 and 14 Day

(SDF1a p=0.10, Saline p=0.06). Uninjured muscles at day 14 showed no significant dif-

ference in fiber composition and thus were combined to compare to the various treated

injury groups. At 14 days, SDf1a and saline muscle groups showed significant changes

compared to uninjured muscle, including decreased stable fibers (SDF1a p=0.0000001,

saline p=0.0000002), increased regenerating fibers (SDF1a p=0.00001, saline

p=0.000006), and increased degenerating fibers (SDF1a p=0.033, saline p=0.00001)

When comparing SDF1a treated injured muscle with saline treated injured mus-

cles the morphology data at day 7 and day 14 showed no significant differences in stable

or regenerating fibers. At day 7 there was no significant difference in degenerating fibers

as well. However, at day 14 it was found that SDF1a treated injured muscle tissue had a

significantly lower mean degenerating muscle fiber count than saline treated injured

muscle tissue (p=0.03). Muscle composition showed overall healthier muscle tissue in

SDf1a treated animals by virtue of smaller percentage of degenerative and larger percent-

age of both regenerative fibers, and stable fibers as compared to saline (Fig 13.)

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Fig 14. H+E examination of injured muscle tissue. A. Mean number of fibers. Muscle fibers delineated by the following criterion. Central nuclei > 3 Degenerative, Central Nuclei <3 Regenerative, No central nuclei Stable. Significance **p=0.01, * p=0.05.

�47

Fig 15.%Composition of muscle fibers, total compared to individual categories of muscle fibers.

Histologic Analysis: Pax 7 Immunohistochemistry

Pax7 is a myogenic marker of early regeneration and typifies a muscle progenitor

cell also known as a satellite cell. These cells have been postulated to be necessary for

recovery from muscle injury [76], [77]. Pax7 immunohistochemistry was performed to

quantify populations of these cells in the injured tissue. 8um tissue was sectioned from

injured animal muscle tissue and stained to expose pax7 nuclei. Results are depicted in

figure 17 but in short I found that at both days 7 and 14 the number of pax7 positive nu-

clei per high powered field were significantly higher in the SDF1a treated group when

compared to Saline (p <0.05). The mean number of positive nuclei per high powered field

also significantly decreased when examining SDF1a treated muscles at 7 days and 14

days (p<0.01).

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Fig 16. Pax7 Immunohistochemistry. Mean number of nuclei counted through ImageJ processing. ** significant to p=0.01

Molecular Analysis: 7 and 14 Day Muscle Regenerative QPCR

Muscle regeneration is a complex process but has distinct markers determining

what stage of regeneration a muscle fiber is at. Pax7 elucidates early regeneration and

quiescent stem cell populations. MyoD is a downstream marker of muscle formation, and

myogenin (MyoG) is a sign of late tubule formation and integration into the muscular

unit. I performed PCR evaluating these markers to understand SDF1a’s effect on muscle

regeneration. All QPCR results are compared to house keeping gene GAPDH to deter-

mine relative expression. Results are depicted in Figure 18 but in summary at day 7 and

day 14 QPCR showed in SDF1a treated muscles there was a greater expression of Pax7

and MyoD though these were not found to be statistically significant when compared to

Saline treatment (Day 7 p= 0.39, p= 0.83; Day 14 p=0.29, p=0.718). MyoG was found to

less expressed in SDF1a treated animals as compared to saline at both day 7 and day 14

which, again, was not found to be statistically significant (Day 7: p=0.91; Day 14:

p=0.92).

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Fig 17. Muscle Regenerative QPCR. A. Day 7 markers Pax7, MyoD, and MyoG. B. Day 14 markers pax7, MyoD, MyoG

Histologic Analysis: Trichrome Blood Vessel Analysis:

Trichrome staining was performed on 8um sectioned slides. At least 5 muscle im-

ages per animal were taken and analyzed through ImageJ cell counter and measuring

tools. Vessel size was measured from greatest diameter across tunica adventitia. Typical-

ly uninjured muscle fibers have a low number of vessels per high powered field, and ves-

sel size is typified by large arterioles, or very small capillaries. Reactive vasculature tends

to be in an intermediate/small size. Results are depicted in figure 19 but in summary It

was found that there were significantly less vessel numbers per high powered field at day

7 (p=0.05), though this was not seen at day 14 (p=0.96). Average vessel size was larger in

the SDF1a treated animals at day 7 but smaller at 14 days when compared to Saline treat-

ed animals. However vessel size was not found to be significant when comparing the

treatment groups (p=0.63).

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Fig 18. Trichrome blood vessel analysis. All images scaled from 1600x1200 pixels using ImageJ measurements. A. 7&14 day Blood vessel number counted significance of **p=0.01. B. 7&14 day Blood vessel size measured from largest diameter across tunica adventitia

Discussions The CS model that is being studied causes multifactorial tissue damage including

disruptions to vascularity, nervous tissue, and muscle architecture [1] [76]. The environ-

ment produced by injury is also complicated by infiltration of inflammatory cells, and

the resultant fibrosis. The ultimate test of skeletal muscle health is in its ability to create a

contractile force and a return to baseline muscle function is the ultimate test of the recov-

ery from muscular injury. Muscle function can then be utilized as a birds eye view of the

overall health of a muscle. I hypothesized that an increase in the regenerative capabilities,

either through utilizing the angiogenic protein VEGF, or the stem cell recruiting abilities

of SDF1a would allow for a tipping of the scales towards regeneration in the injury envi-

ronment and a return to baseline muscle function. Our results showed that in regards to

muscle function the effects of VEGF had a more pronounced, though delayed, influence,

showing greater efficacy between the time points of day 7 and day 14 as compared to

saline (control). Muscle function was less impacted by SDF1a and the honing of stem

cells to the site of injury at the time points studied. Our microscopic and molecular re-

sults obtained through these experiments indicated that the biologic therapy we utilized

did have an effect on the CS environment. The results gathered can be used as ground-

workings for further study determine which direction future experiments should proceed

�54

to maximize regeneration and a ultimately reach a return to baseline function in the CS

model.

VEGF Experiments:

The significant findings in the VEGF experiment included a significant increase

in mean stable muscle fibers, blood vessel numbers per high powered field, and muscle

function between day 14 and day 7. Also noted was a significant decrease in mean degen-

erating muscle fibers. In essence VEGF utility seemed to be in the effect on muscle

fibers, vascularity, and muscle function which also includes nervous involvement. These

aspects of VEGF involvement are explored further.

VEGF effect on vascularity

The effects of VEGF on vascularity are well defined and delineated through em-

bryogenic and tumorigenic models [78]. A practical way to address ischemic muscle in-

jury is with VEGF supplementation given it’s well delineated natural vessel growth ef-

fects. Vessel damage is one of the hallmarks of the CS model [.76]. In our experiment

VEGF treated muscles showed a significant increase in vessel number per high powered

field at day 14 as well as an increase in overall muscle functional regeneration between

days 7 and 14 when compared to saline treated controls . Takeshita et al also found simi-

lar results in a chronic ischemia model with VEGF therapy, noting specifically a higher

angiographic score after a single intramuscular injection of VEGF, and a significant in-

crease in blood pressure in the ischemic muscle when compared to no treatment. How-

�55

ever, no muscle function was tested overtly [53]. In the Takeshita et al experiment the

action of VEGF was particularly evident at day 10, a time point which correlates with the

functional recovery we noted. This could indicate that the VEGF induced maturity of

vasculature begins around this time, given the increases in blood pressure noted in the

Takeshita et al experiment . Further expounding on the role of vasculature in recovery

from ischemic injury Hopkins et al found that in a rabbit model, sustained, controlled,

low dose release of VEGF was linked with significantly enhanced neovascularization.

The degree of vascular perfusion and morphological appearance of musculature after

treatment were near normal, and exhibited similarly normal function. The sustained re-

lease model showed these promising results at 14 days.

In our study, although vessels were more numerous on average in VEGF treated

injured muscles, this in no way speaks to the patency of said vessels. Bauters et al for

example showed that peak flow in an ischemic hindlimb, when supplemented with in-

traarterial VEGF, was not significantly increased compared to control until day 30 [79].

Given these conflicting results as to the true nature of vessel maturation under the influ-

ence of VEGF, A way to further analyze the vessel architecture is therefore needed to

clarify the results obtained in our experiment. This can be accomplished through imaging

modalities such as microCT, to determine the degree of functionality of the vessels influ-

enced by VEGF. Ultimately, this will provide a better understanding of the role of vascu-

larity in the recovery of muscle function in the CS model.

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VEGF effect on Muscle Progenitor Cells

Muscle function is dependent on contractile muscle fiber units, adequate neuronal

stimulus, and the ability to perform contractions using cellular respiration for energy.

These tenets are essential for a muscle to function correctly, and are all effected in the CS

model. Our experiments on VEGF supplementation in the CS hindlimb has shown to sig-

nificantly increase the number of blood vessels per high powered field compared to

saline, but the ability of the muscle studied to form contractile units is dependent on more

than blood supply. Functional recovery also requires an adequate supply of muscle

progenitor cells , satellite cells, to recover from damage [24]. The ability for muscle

progenitor cells to survive in the ischemic environment indicative of CS is also essential

[80]. The link between vascularity and muscle development has been studied showing

many interesting results. First it was found that when VEGF was expressed as a vector in

an ischemic hindlimb model, there was an increased number of regenerative cell mor-

phology in the injured muscle, indicated by central nuclei. This action of VEGF led to

further study into receptor expression on muscle progenitor cells (MPC), what we have

referred to previously as satellite cells. This study elucidated both VEGFR1 and VEG-

FR2 were present on the surface of MPC’s[64],[67]. Although these cells are responsive

to VEGF, in our study we noted that when VEGF was supplemented to the ischemic

hindlimb there was no significant increase in muscle progenitor cell nuclei myogenic fac-

tor pax7 on immunohistochemistry, or in expression of myogenic markers. However,

morphological data could explain this discrepancy as it was seen that there were a signifi-

cant number of stable fibers, and significant decrease of degenerating fibers. This could

�57

indicate that the action of VEGF on muscle cells may be less involved in chemotactic ac-

tivity and more in cell survival. Another reason for this can be time point, as pax7 is an

early marker but only one in a a continuum of myogenic markers. The expression profile

showed a greater expression, though not significant, of many of these transcripts

(pax7,MyoD, and MyoG) and could have been a case of a study end point which did not

capture the periods where each is maximally expressed [67]. Another potential reason

for the differing results is the duration of effect in VEGF treatment used in our experi-

mental model. We used a bolus delivery with 3 time points, where as in other studies, in-

cluding the AAV vector delivery of Bauters et al, a consistent secretion of VEGF was

utilized to mitigate damage in the ischemic hind limb model [81]. This allowed for con-

tinuous action of VEGF in the ischemic environment and a potentially beneficial effect

on muscle progenitor differentiation [64]. Adopting a continuous release or expression

strategy can allow for a greater understanding of muscle cell function when VEGF is

supplemented to the CS environment.

VEGF effect on Nervous tissue

Along with regenerative muscle progenitor cells, having the nervous tissue to simulate

muscle function is integral for the maintenance of healthy muscle. Post injury, these

neural networks are disrupted as confirmed by the model of CS [76]. Part of the multifac-

torial treatment effect of VEGF is the ability to direct neurogenesis. Sondell et al showed

that increased regenerative potential was acheived through VEGF binding to flk1 recep-

tors in explanted cells of the the dorsal root ganglion (DRG) and superior cervical gan-

�58

glion (SCG). When 100 ng/ml of VEGF was applied to these explanted cells, VEGF was

shown to bind to schwann cells eventually leading to axonal outgrowth in these zones

[65]. In vivo studies using a VEGF secreting osmotic pump showed increased neurogene-

sis and not the typical decrease in cell death expected in the neuroproliferative subgranu-

lar zone and the subventricular zone of the mouse brain. These and other studies show the

neurotropic effect of VEGF are a potential mechanism of action in increasing muscle

function in the CS model and is a promising area of further study into these effects [82]

[83] [84].

SDF 1a experiments

Stromal cell derived factor 1 alpha (SDF1a) has shown a variety of effects in rat

CS muscle, among these are significantly increased numbers of pax7 positive nuclei per

high powered field, decreased fibrosis and degenerating fibers at day 14 compared to

saline, and no significant difference in stable fibers when compared to uninjured muscle

at day 7. These effects however did not produce a return to baseline muscle function at

day 7 and day 14. SDF1a injured muscle function results from twitch to tetany were not

significantly different when compared to saline injured muscle function. These results

can be potentially explained through looking at the kinds of cells SDF1a recruits to the

areas in which its concentration had been externally supplemented.

SDF1a and Muscle progenitor cells

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SDf1a activity was originally thought to be limited to hematopoietic stem cells

and has well delineated honing and adherence effects on these cells in vitro and in vivo.

The fidelity of SDF1a to its receptor CXCR4 allows for easy study into which cells are

responsive to SDF1a in vitro and in vivo. Among the cells that express CXCR4 are satel-

lite, pax7 positive cells[85] . These muscle progenitor cells provide the raw regenerative

material for recovery from muscular injury[24, 86]. Honing to sites of injury is a potential

way to increase the efficacy of this process. Our results showed that although SDF1a

treatments led to a significantly increased pax7 positive nuclei present in the muscles ef-

fected by CS damage, this did not correlate with a return to muscle function. Previous

studies including Bzoska et al, had previously analyzed muscle health in terms of percent

make up of stable fibers, regenerating fibers, and muscle mass as compared to no inter-

vention [55]. These are used as analogs to muscle health, however the parameters did not

include muscle function. When muscle function was performed in our experiment, though

the make up of our muscle tended to resemble healthy muscle, our results did not show

recovery of function that would speak to regeneration. Understanding more about ner-

vous architecture in these tissues is then an important point for further study, as it is a key

missing piece in the interpretation of functional data. The role of pax7 use in muscle

function recovery has been studied through multiple in vitro and in vivo studies [24].

However, studies into SDF1a have shown that MPCs are not the only cells which can

participate in recovery from muscle damage. In a study by Kowalski et al, pax 7 -/- mice

which were injured through cardiotoxin hindlimb injection and treated with SDF1a and

GCSF injections showed a return to stable muscle architecture, lower creatinine kinase

�60

levels, and lower fibrosis levels as opposed to wild type no intervention injured mice [87]

suggesting that other non-MPC cells can participate in the attenuation of muscular

injury . Though again muscle function was not studied to determine the ability of these

cells to form contractile units. These studies show that pax7 positivity may not be the

only factor at work in SDF1a induced muscle recovery, and further study would do well

to characterize the cells active in the SDF1a treated injury muscle and how muscle archi-

tecture is able to be maintained in the absence of pax7 cells.

SDf1a effect on inflammation and fibrosis.

One of the more robust significant findings in the SDF1a experiments was the de-

crease in fibrosis seen at day 7 and day 14. Fibrosis typically occurs after injury where

macrophage infiltration leads to recruitment of fibroblast and resultant fibrotic scarring.

[72]This process is mediated by the two types of macrophages M1 and M2, as well as

fibroblast response to pro-fibrotic cytokines such as TGF-B [72]. Theory would dictate

that because SDF1a is a hematopoietic stem cell recruiter, which among this family exists

fibroblasts, that SDF1a would be a pro-fibrotic peptide. However studies into SDF1a fi-

brotic effects are inconclusive. Brzoska et al has showed that fibrosis in SDF1a treated

muscles which underwent hindlimb denervation and crush injury, and received SDF1a

injections at the time of injury, showed significantly less fibrosis than control muscles

[55] . The effect of SDF1a in reducing muscle fibrosis is less studied though other models

of SDF1a’s role in fibrosis are more fleshed out. In a bleomycin induced pulmonary fi-

brosis model early antagonism of the CXCR4/CXCL12 axis with AMD3100 showed in-

creased fibrosis potentially owing to the increase in early inflammatory response and in-

�61

crease in vascular leak[88, 89]. Opposing results were noted in subsequent studies where

the result of SDF1a axis block was found to have significantly less fibrosis [90, 91]. In

hepatic injury models fibrosis was increased with inhibition of SDF1a signaling cascade

owing to a detrimental modulation of the inflammatory response favoring neutrophil in-

filtration [92]. In our study utilizing VEGF for treatment, we had noted that VEGF sup-

plementation had increased fibrosis when compared to saline. VEGF is notorious for

extravasation which could suggest that fibrosis is in part a byproduct of this leak and that

timing of SDF1a administration is the difference between significant inflammatory in-

volvement and resolution of fibrosis.

ConclusionsFuture experiments

Improving the current model

Our model of CS in adult rats achieves the goal of multifactorial injury, including

disruptions of vasculature, nervous architecture, ischemic muscle damage and fibrosis.

Only minor adjustments are needed in this protocol and include a more uniform degree of

injury in rats and a more reliable way to induce this injury, other than manual mainte-

nance of neonatal sphygmomanometer pressure.

The timing and method of treatment delivery is another way to improve the cur-

rent model. Timing of therapy has practical utility given the well delineated, step-wise,

nature of skeletal muscle regeneration. The first stage of skeletal muscle regeneration,

known as the destruction/inflammatory phase, is characterized by rupture of myofibers

�62

and inflammatory cell reactions. The cells active in this stage typically involve neu-

trophils and monocytes[93, 94]. The second stage of repair is characterized by phagocy-

tosis of necrotic muscle, regeneration of new muscle fibers, and the beginning of scar

formation. This stage is predominantly macrophage and fibroblast dependent, and is

when satellite cells begin to differentiate into myoblasts [94] [93]. The final stage is the

remodeling phase, where the ground work from the second phase will either lead to pre-

dominant scar formation, fibrosis, or recovery of muscle tissue [94] [93]. During these

phases a variety of effects of biologics can be exerted, especially with the biologics we

are studying, SDF1a and VEGF. SDF1a’s ability to act as a recruitment factor for

hematopoietic cells can increase the inflammatory reaction as well as increase the regen-

erative material. VEGF’s ability to both act as an angiogenic factor and a permeability

factor leads to similar issues regarding increasing inflammatory reaction as well as regen-

eration of vessels. Testing supplementation through evaluating the stage specific effects

of biologics on skeletal muscle regeneration is a way to increase the efficiacy of the

treatment model. Increasing the fidelity of treatment administration to the stages of re-

generation where they are most effective is a practical way to increase the efficacy of our

therapies.

Furthermore, kinetics of current biologic therapy can be studied more efficiently

through a continuous sustained release method (vector, osmotic pump) which has been

shown to have differing effects than the bolus supplementation method [ 95] [96] . Tim-

ing and duration of therapy are two ways to alter the current model to further evaluate the

efficacy of biologic use.

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Further clarification of results.

A few results which could be further clarified in our experiment include, deter-

mining not only the number of vessels but the architecture of said vessels in the VEGF

experiment, and the origin of cell types noted in the SDF1a experiment.

In the VEGF experiment, we used trichrome staining and Image J processing to

count the number of vessels in injured muscles per high powered field. This gives limited

insight however into what the architecture of these vessels are. It is thereby important to

understand if these vessels are organized into functional units, or disorganized into patho-

logic units such as capillary hemangioma. Vessel architecture is best studied through pre-

injury microCT evaluation of muscle vasculature, as well as post-injury, prior to tissue

collection, evaluation of treatment induced changes to the injured vasculature.

In regards to the SDF1a experiment, a determination of the origin of stem cells

involved in therapy could be used to increase the validity of the conclusions drawn. This

can be accomplished through radioablating bone marrow cells, and replacing with GFP

labeled marrow cells. This is important given the results from Kowalski et al that muscle

regeneration is not entirely dependent on Pax7 when SDF1a treatment is initiated [87]

Fluorescent labeling of marrow stem cells will allow us to understand if circulating mar-

row origin hematopoetic stem cells are involved in the therapeutic action of this

biologic. .

Improving Treatment Delivery.

Biomaterial biologic delivery has been a recent trend in maximizing the efficacy of and

targeted delivery to areas of injury [96] . In our model this has practical advantages. First

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as an implant it will spare the already injured musculature from repeated intramuscular

injections in the case of VEGF, and the single injection in the case of SDF1a. Second, the

secretion profile can be accurately modeled through in vitro studies so it will be possible

to understand the amount of drug delivered per unit of time. Third the ability to co-seed

biologics can speak to synergistic effects of these biologics in the CS model. For in-

stance, the co-administration of VEGF and SDF1a has shown to produce a greater degree

of therapeutic angiogenesis in an ischemic hindlimb model than either factor alone [58].

A downside to the addition of a biomaterial to the CS environment has to do with

the further increase in inter-compartmental pressure. Bulky biomaterial treatment has the

potential to further decrease the level of venous outflow leading to an exacerbation of

damage. This potential drawback can be mitigated through the usage of less bulky bioma-

terials such as PLGA. Biomaterial delivery of biologics is another way to increase the

efficacy of treatment in the CS model.

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CVNeel Sharma

nesharma@wakehealth.edu neelsharmamd@gmail.com

(304)-280-4694 Permanent Address 66799 Anna Dr. St. Clairsville, OH 43950 Local Address 2452 Winterwoods Lane Apt#206. Winston-Salem, NC 27103

Education Wake Forest Institute for Regenerative Medicine-Molecular Medicine and Translational Sciences Masters of Molecular Biology (anticipated) 2015-2017

Internal Medicine Resident Wake Forest Baptist Medical Center 2014-2015.

Northeast Ohio Medical University, Rootstown OH MD, May, 2014

Kent State University, Kent OH Integrated Life Sciences, May 2010. Magna Cum Laude Presidents List

Research & Employment Experience Wake Forest Institute for Regenerative Medicine • “Vehicles in cellular therapy, host response chapter submitted.” Submitted for publishing. • “Maxillary hypoplasia incidence in Isolated Cleft palate repair: Akron Children’s Experience”

Currently in review. • “Adipose Stem Cells: A Surgical Perspective” Poster presented at Wake Forest Baptist Medical

Center Surgical Resident and Fellow Research Symposium. • “GDF11 treatment has no effect on the recovery of skeletal muscle function in a rat model of

compartment syndrome injury.” Submitted for Review Tissue engineering and regenerative medicine, Co-author.

• “An evaluation of intramuscular VEGF in an aged murine model of post-acute compartment syndrome” Presented at WFIRM Molecular medicine and Translational Science Seminar, sub-mitted for TERMIS AM annual conference poster presentation.

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Cleveland Clinic Research Assistant, Pulmonology, Cleveland Clinic, Cleveland, OH. Summer 2011 "Evaluation of traffic related air pollution exposure estimates in asthmatics using land use regres-sion during the Cleveland Multiple Air Pollutant Study" Principle Investigator: Sumita Khatri, MD • National Institute of Health T32 grant recipient for Summer Research. • Analyzed Population Heat maps • Input data on Asthma prevalence • Ran statistical regression based on data collected • Used GIS software for understanding the spatial distribution of patients with asthma exacerba-

tions. • Poster presenter at the 9th Annual AMA Medical Student Section and Resident and Fellow

Section Research Symposium in New Orleans, Louisianna.

Kent State University Research Assistant, Aquatic Microbiology, Kent State University, Kent, OH. Summer 2010 Principle Investigator: Laura Leff, PhD • Collected Samples • Assisted in Lab • Observed Analysis • Assisted in thesis preparation

GameStop St. Clairsville OH July 2007-January 2008 www.gamestop.com • Game Advisor

Leadership & Activites

Student Interest Group in Neurology and Neurosurgery 2010-2014 President 2011-2012 • Organized Fundraising chocolate pretzel sale • Set up a group meeting with a clinical Neurologist at a local restaurant • Coordinated a trip to the American Academy of Neurology Conference in New Orleans LA • Organized a M4 panel to discuss experiences in the National Residency Match. • Manned the most popular event at the NEOMED public health fair, displaying human brains

to the people of Rootstown.

I Love Ice Cream Club 2010-2012 President 2011-2012 • Catered a Senior citizen Halloween party community service event with the Geriatrics Interest

group.

Future Entrepreneurs of America 2010-2011 Secretary and Treasurer 2010-2011

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American Medical Association 2010-present Attended an AMA conference at the University of Cincinnati and: • Volunteered at a community health fair checking blood pressure, pulse, respiration rates, glu-

cose levels, and general health • Participated in a seminar on underserved care • Learned about new robotic surgery methods and operated the DaVinci robot.

American Academy of Neurology 2010-2014 Attended Annual meeting in New Orleans, Louisianna

Service

Hartville Migrant Clinic, Hartville, OH June 2011 • Administered Tetanus Shots and PPD skin tests for Tuberculosis.

Volunteered at the Open M free Clinic, in Akron OH. April 2011 • Filled prescription orders and assisted in script writing to be signed by physicians

Bhangra Dance Competition at The University of Buffalo. Buffalo, NY, 10/29/2011 • Choreographed and participated in a collegiate dance competition

Pitcher for the Akron General Medical Softball Team Summer 2010 • Summer league

Tournament Judge at Fairmont State University Speech and Debate Tournament January 2010

Played Piano and Violin for many years • Attained a Superior ranking for piano

Summer Camp Counselor, Camp Lambec, Erie PA August 2009

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