Materials & Methods

Main Results: Behavior

Program # 139.23Poster #

Treatment of repetitive, moderate traumatic brain injury with a highly selective, orallyactive arginine vasopressin V1a receptor antagonist.

*N. G. SIMON1,2, T. MORRISON3, P. KULKARNI3, X.Cai3, S.-F. LU1, A. SIDWELL2, S. O'NEAL2, C. F. FERRIS3

1Azevan Pharmaceuticals, Bethlehem, PA; 2 Dept. of Biological Sciences, Lehigh University, Bethlehem, PA 3Center for Translational Neuroimaging, Northeastern University, Boston, MA

Resting State Functional Connectivity

Imaging Neuroanatomy

Experimental Design

This This TThis work was supported by Azevan Pharmaceuticals

IntroductionArginine vasopressin (AVP) is a chemical signal in the brain influencing cerebral vascular resistance and

brain water permeability and contributes to the pathophysiology of brain edema following head trauma. These cerebrovascular effects are mediated through the AVP V1a receptor, which is highly expressed in cortical and subcortical brain areas across all mammals.

To study the therapeutic potential selective V1a receptor antagonism on moderate TBI, we adopted the momentum exchange model developed by Viano and colleagues. This model features several translational advantages over other head injury models. For example, the head, neck and body can move with impact, and the velocity of head movement and energy transfer can be calculated and scaled for mild, moderate, or severe concussions. These studies were conducted with moderate impacts with neuroradiological evidence of contusions. Male rats were concussed twice with one day between. Approximately 24 hrs after the first concussion, rats were given vehicle or AVN999, a highly selective V1a receptor antagonist from AzevanPharmaceuticals that crosses the blood brain barrier. Sham non-concussed rats were given the V1a antagonist alone. Rats were treated twice daily for 5 consecutive days. At two weeks post-concussion, cognitive (Barnes maze, novel object recognition) and motor (beam walk and rotarod) behaviors were assessed and neuroradiological MRI performed. Concussed, untreated rats showed significant deficits in cognitive behavior. Rats concussed and treated with the AVN999 showed no deficits in learning and memory and were no different from sham controls. There were no significant differences in motor behavior between groups. Concussed/drug treated rats showed enhanced functional connectivity in hippocampal circuitry and a reduction in injury-evoked edema near the site of impact.

T2 Relaxometry

Summary

Speculation

Main Results: Imaging

Resting State Functional Connectivity Edema and Ventricular Volumes

Fig 1 Fig 2

Fig 3 Fig 4

Fig 3 Correlation matrices of 166 rat brain areas for rsFC comparing TBI to sham controls (top left) and TBI to AVN999 treatment. The brain areas with significant correlations often appear as clusters because they are contiguous in their neuroanatomy and function. The diagonal line separates the different experimental groups. The pixels locations are mirror images. The marked areas labelled with letters A-N show highlight clusters that are similar and in many cases very different between experimental groups (see Fig 3 Legend). The ventral hippocampus shows hyperconnectivity following TBI + AVN999 treatment. The connections are shown in the table. The areas in dark blue comprise the key nodes of the ventral hippocampus. In sham controls, these nodes have functional connections with the adjacent amygdala (light blue), limbic cortex (yellow), and reticular n. of the midbrain (red), a key component of the reticular activity system necessary for arousal and attention. The 3D organization of these brain areas are shown in the glass brains below.

Fig 4 The volume of lateral ventricles at the level of impact (hippocampal fimbria/septum) was calculated and compared using a single tailed T-test. There was no significant difference between sham and TBI + AVN999 groups while the volume in the hit alone group was significantly greater than sham and drug treatment

Fig 1 Rats in the HIT condition spent significantly less time, compared to chance (i.e., 50% of total object investigation) investigating the novel object (t(5) = 3.393, p < 0.05), while both the DRUG (t(6) = 3.08, p < 0.05) and the DRUG+HIT (t(5) = 2.61, p < 0.05) groups spent significantly greater than a chance amounts of time with the novel object.Fig 2 One-way ANOVA and followed by Fisher’s protected LSD revealed the HIT group had significantly longer goal box latencies compared to both the DRUG (p < 0.01), and the DRUG+HIT group (p < 0.05), with no differences between the DRUG and the DRUG+HIT group (p > 0.6). When analyzed across testing days, there were main effects of both Testing day (F[3, 48] = 14.89; p < 0.0001), and Condition (F[2, 16] = 5.885, p < 0.01), with no significant interaction (F[6, 48] = 0.78; p > 0.5). There was no difference between the DRUG and DRUG+HIT groups (p >0.8).

At the beginning of each imaging session, a high-resolution anatomical data set was collected using the RARE pulse sequence (20 slice; 1 mm; field of vision [FOV] 3.0 cm; 256 × 256; repetition time [TR] 2.5 sec; echo time [TE] 12.4 msec; NEX 6; 6.5-minute acquisition time)

Images were acquired using a multi-slice multi-echo (MSME) pulse sequence. The echo time (TE) was 11 ms, and 16 echoes were acquired during imaging with a recovery time (TR) of 2500 ms. Images were acquired with a field of view [FOV] 3 cm2, data matrix = 256×256×20 slices, thickness = 1 mm. Values for longitudinal relaxation time (T2) were obtained from all the slices using ParaVison 5.1 software. T2 was used to characterize the edematous volume in the lateral ventricle. The T2 values were used for segmentation and quantification of the ventricle volume The T2 values were computed using the equation; y = A+Cexp (-t/T2) (S.D. weighted) obtained from the Paravision 5.1 software. Where, A = absolute bias, C = signal intensity,t = echo time and T2 = spin-spin relaxation time. The ventricle was identified as a hyperintensity on the T2 map over three 1 mm sections. The volume was calculated using a snake region growth algorithm in itk-SNAP (www.itksnap.org). The threshold was set at 6300 to 9000 as absolute pixel intensity. A point is seeded within the ventricle and the algorithm run until segmentation is complete

LEGEND Fig 3A: HypothalamusB: Dorsal Hippocampus – CA1, CA3, dentate C: Thalamus D: Sensory Motor CTX & Dorsal Striatum – (motor CTX, anterior cingulate, primary SS CTX), dorsal striatum E. Prefrontal CTX & Ventral Striatum – (prelimbic ctx, infralimbic ctx, ventral & lateral orbital), (ventral striatum)F. Amygdala & Piriform/Insular CTX G. Ventral Hippocampus & Temporal CTX - CA1, CA3, dentateH. Cerebellum – All lobes, Crus1&2, medial cerebellar n., (fastigial)I. Reticular Activating System Medulla & Cerebellum – (interpose n., copula, 10th

lobule), (gigantocellularis, parabrachial, parvocellular reticular, solitary tract n. vestibular n., principle sensory n. trigeminal)J. Cerebellum & Medulla & Entorhinal CTX – (paramedian lobule, paraflocculuscerebellum, flocculus), (ventral subiculum, entorhinal ctx, ectorhinal ctx), (median raphe, locus ceruleus, trapezoid body, dorsomedial tegmentum, facial n. sub coeruleus, pontine reticular n. cochlear n. dorsal paragigantocellularis)K. Midbrain Dopamine – substantia nigra compacta, substantia nigra reticularis, ventral tegmentumL. Hypothalamus |connections| AmygdalaM. Reticular Activating System|connections|Cerebellum|connections|Raphe|connections|Hippocampus|Reticular Activating SystemN. Amygdala|connections|MedullaO. Midbrain Dopamine|connections|Ventral HippocampusP. Midbrain Dopamine|connections|Hypothalamus|Hypothalamus

Motor Behavior

Male Sprague Dawley rats (n=19; 250-270g) aged 65 days, were obtained from Charles River (Worcester, MA). Rat were maintained on a 12:12 hour light:dark cycle with a lights on at 0700 hours and allowed access to food and water ad libitum. The protocols used in this study complied with the regulations of the Institutional Animal Care and Use Committee at the Northeastern University.

We replicated the pneumatic pressure drive, 50g compactor described by Viano and colleagues and reproduced consistently the 7.4, 9.3 and 11.2 m/s impact velocities described for mild, moderate, and severe rat head injury, respectively. The data reported here all came from a 9.12 meters/sec impact velocity as determined using high-speed video recordings

Beginning on postnatal (P)70, animals were divided randomly into 3 groups (n = 6-7/ group; DRUG, HIT, and DRUG+HIT) and the head impact and drug regimens were started. All groups were anaesthetized with 4% isoflurane in oxygen, and HIT and DRUG+HIT groups received the first of two head impacts using a custom setup from Animal Imaging Research (AIR; Holden, MA) on P70. The day after the first impact (P71), the AVN999 (or saline for HIT animals) was administered to the DRUG and DRUG+HIT groups twice per day at 0700 and 1400 (IP @10mg/kg). The drug regimen continued over the following 4 days (5 days total). The second head impact was delivered two days following the first on P72. Two weeks post concussion, rats were imaged and tested for cognitive and motor behaviors.

Resting-state fMRI scan were collected using spin-echo triple-shot EPI sequence (imaging parameters: matrix size= 96x96x20, TR/TE=3000/15 msec, voxel size=0.312x0.312x0.12mm, slice thickness= 1mm. Preprocessing in this study was accomplished by combining Analysis of Functional NeuroImages(AFNI_17.1.12, http://afni.nimh.nih.gov/afni/), FMRIB Software library (FSL, v5.0.9, http://fsl.fmrib.ox.ac.uk/fsl/), Deformable Registration via Attribute Matching and Mutual-Saliency Weighting (DRAMMS 1.4.1, https://www.cbica.upenn.edu/sbia/software/dramms/index.html) and MATLAB (Mathworks, Natick, MA). Brain tissue masks for resting-state functional images were manually drawn using 3DSlicer (https://www.slicer.org/) and applied for skull-stripping. Normalization was completed by registering functional data to the MRI Rat Brain Template (Ekam Solutions LLC, Boston, MA) using affine registration through DRAMMS. The region-to-region functional connectivity method was performed in this study to measure the correlations in spontaneous BOLD fluctuations. A network is comprised of nodes and edges; nodes being the brain region of interest (ROI) and edges being the connections between regions. 171 nodes were defined using the ROIs segmented from our custom MRI RAT Brain Atlas. Voxel time series data were averaged in each node based on the residual images using the nuisance regression procedure. Pearson’s correlation coefficients across all pairs of nodes (14535 pairs) were computed for each subject among all three groups to assess the interregional temporal correlations. The r-values (ranging from -1 to 1) were z-transformed using the Fisher’s Z transform to improve normality. 171 x 171 symmetric connectivity matrices were constructed with each entry representing the strength of edge. Group-level analysis was performed to look at the functional connectivity in controls, one hit and three hit groups. The resulting Z-score matrices from one-group t-tests were clustered using the K-nearest neighbors clustering method to identify how nodes cluster together and form resting state networks. A Z-score threshold of |Z|=2.3 was applied to remove spurious or weak node connections for visualization purposes.

• Moderate traumatic brain injury using a momentum exchange model produced clear neuroradiological evidence of contusions.

• The injury to the brain in untreated rats caused significant deficits in learning and memory.

• Injury to the brain in untreated rats was associated with enlarged lateral ventricular volumes indicative of enhanced inflammation and edema.

• Injury to the brain in untreated rats resulted in hypoconnectivity in hippocampal neurocircuitry.

• Treatment within 24hrs after the first concussion with a selective V1a receptor antagonist prevented the cognitive problems, resulting in behavioral measures no different from sham controls.

• V1a receptor antagonist treatment reduced the edematous enlargement of the lateral ventricles.

• V1a receptor antagonist promoted hyperconnectivityin hippocampal neurocircuitry.

The data clearly show that a V1a receptor antagonist that crosses the BBB can treat the cognitive and neurobiological effects of moderate brain injury. The mechanisms may be two-fold: 1) reducing the inflammation and edema caused by the head injury and, 2) promoting neuroadaptive changes in functional connectivity to compensate for the trauma.