10 neurochemistry of human postmortem brain

Neurochemistry of Human Postmortem Brain

Gavin l? Reynolds and C/are f. Beasley

1. Introduction

The use of human postmortem brain tissue in neurochemical and neuropharmacological research has received increasing attention over recent years. In fact, there is one work that, more than any other, can be identified as being responsible for the interest in this approach. It was Birkmayer and Hornykiewicz who, having observed a deficit in the content of the neurotransmitter dopamme m brain tissue taken postmortem from patients with Parkmson’s disease, set about to counteract this deficit in living patients by treatment with L-dopa. The identification of an abnor- mally low transmitter concentration and its supplementation by the administration of the appropriate biochemical precursor has revolutionized the treatment of this disease (Ehringer and Hornykiewicz, 1960; Birkmayer and Hornykiewicz, 1961). It has also served to motivate neurochemists to study other neurological and psychiatric diseases using postmortem brain tissue. Such a success story has yet to be repeated. Nevertheless, neurochemical and other molecular approaches to studying the diseased brain have generated a large amount of information that has increased enormously our understanding of the molecular neuropathology and neural dysfunction m these disorders.

This defines one major goal for studies involvmg postmortem human brain tissue: to determine the molecular and cellular pathology of diseases of the human brain, the neurological and psychiatric disorders. A further aim is to understand the actions of drugs that act on the brain to relieve, or induce, such disorders. With this, and with further information from other more basic

From Neuromethods, vol 33 Cell Neurobiology Techmques Ed A A Boulton, G I3 Baker, and A N Bateson 0 Humana Press Inc

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320 Reynolds and Beasley

biochemical studies, it is hoped to add to our picture of the complex integration of cellular and molecular processes that we call human brain function. This chapter aims to provide an overview, with examples taken from work in the authors’ laboratory and elsewhere, of the use, values, and potential of studies employing human brain tissue taken at autopsy.

2. Human Brain or the Animal “Model”

The small mammal bred for laboratory use has many undeni- able advantages over human tissue taken postmortem. Severe neu- rosurgical procedures, acute or chronic drug treatment, and more subtle manipulations such as dietary adjustments or behavioral trainmg, can precede neurochemical or histological investigation m these animals. Nevertheless, such experiments can be mislead- ing. The importance of results from animal studies is often over- emphasized, and it is very tempting to draw analogies with human brain function when faced with interesting results from experiments performed on the rat. The danger of such extrapolation is understood by every good scientist and yet, all too often, may be overlooked or ignored. There are innumerable examples of metabolic differences between Homo supiens and experimental animals; one very relevant to the study of dopamine function is the relative amount of this transmitter oxidized to dihydroxyphenylacetic acid, about 7040% in small rodents, but only lo-20% in humans, where the major metabolite of dopamine is homovanillic acid. The neurochemical anatomy of the dopamine system is also very different the rat has a mesocortical projection that concentrates on the prefrontal cortex; in humans the cortical dopaminerglc innervation is much more diffuse and distributed throughout the cerebral cortex. Similarly, the enzymes responsible for conjugation and removal of endogenous and exogenous compounds vary greatly between species (and even between races in humans). Such effects will be responsible for species differences in pharmacokinetics and drug disposition. Even in vitro pharmacological studies may dem- onstrate such differences, owing perhaps to variations in antagonist affinity for a receptor or inhibition of a particular enzyme, reflecting subtle differences between species in primary structure of these proteins.

In addition to the direct investigation of brain disease, and avoiding the problem of species differences in metabolic or

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pharmacological study, postmortem analyses may provide other unique opportunities to understand human brain function. The investigation of particular individual groups can prove enlight- ening; one example is the study of patients who underwent leucotomy for the supposed relief of schizophrenia. The neurochemical consequences of this severe iatrogenic lesion of the frontal cortex have been studied (Cutts and Reynolds, 1993). Even individual cases can prove instructive; a patient at risk of Huntington’s disease but dying prior to the onset of symptoms has provided valuable information about the neurochemical and pathological course of the disease (Carrasco and Mukherji, 1986; Reynolds and Pearson, 1990).

2.1. Human Neuropharmacology

Human brain tissue obtained at autopsy is still a very much underused resource for neuroscientific studies. Nevertheless, the past decade or so has seen an increasing recognition of the value of postmortem tissue in neuropharmacological investigation. Simple studies with human tissue often can serve to correct wide- spread misconceptions as to the pharmacological action or speci- ficity of neuroactive drugs (e.g., Reynolds et al., 1982). Not only are the results more relevant to the use of drugs, human tissue is so much more abundant. One brain can provide 10 g of caudate nucleus tissue; over 100 rats would typically be required for an equivalent amount of striatum!

In addition to this straightforward in vitro human neuropharmacology, the effects of previously administered drugs can be studied. Here too individual cases and specific groups of patients can provides valuable information, a key example being the elevation in striatal dopamine D2 receptors in schizophrenia that is apparently associated with prior treatment with antipsychotic drugs (discussed in Subheading 5.3.).

There are probably two major factors that inhibit the use of post mortem brain tissue in pharmacological studies. One is the availability of such tissue; this can often be easily addressed by researchers in medical schools but is more difficult for those in the pharmaceutical industry. The other factor is the common mis- apprehension that post mortem tissue degradation precludes useful biochemical and pharmacological study. Slowly this view, discussed specifically in Subheading 5.5., is being corrected as


valuable and relevant results accumulate in the scientific litera- ture.

3. The Collection and Dissection of Human Brain Postmortem

Some components of the functioning brain are irreversibly dam- aged within minutes after death, and this fact has lead some sci- entists to believe that postmortem material could not be suitable for basic research or studies of diseased brain. However, an increasing number of studies indicate that, when proper precautions are taken, this need not be the case. Unfortunately, there has been a continuous decline in autopsy rate in recent years (Ander- son and Hill, 1989). There is, therefore, a need to standardize procedures for collecting and distributing such human postmortem material that will be acceptable for the research needs of the scientific community. It is important first to pool resources, and this is now being recognized with specialized brain banks, supported by national or international research organizations or charitable foundations, distributing material to researchers all over the world. Second, collaborations between neuroscientists and pathologists must be undertaken to provide samples and information that ful- fill the numerous and differing criteria needed for the wide variety of postmortem studies undertaken in different research groups. These approaches will inevitably require a variety of techniques for preparation of fixed or frozen brain tissue, and may also require CSF and blood samples, information on diagnosis, details of premortem clinical investigations of control and experimental subjects (e.g., psychometric tests), medication, cause of death and agonal state, and gross anatomical and pathological assessments. Nevertheless, recognizing that an increased value is obtained from studies which are comparable between research groups, various national or international bodies have instituted minimal opera- tional criteria for the diagnosis, pathological assessment, and/or preparation of human brain tissue samples. Thus, the (U.K.) Medi- cal Research Council has provided such guidelines for studies of Alzheimer’s disease (Wilcock et al., 19891, whereas a European Union collaboration has published diagnostic criteria for the postmortem study of schizophrenia and affective disorders (Riederer et al., 1995).

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Although the importance of standardizing collection procedures is now slowly being recognized, at present there is still a need to use archival material. This tissue may have been fixed or frozen in such a manner so that it is not suitable for many of the sophis- ticated techniques now employed in modern laboratories; although, in many cases, this tissue can be used for biochemical or histological studies.

Any description of what is involved in the collection of material for studies on human postmortem brain tissue will inevitably represent a compromise between a range of factors. Obviously, not every procedure will be suitable in all situations, and the neuroscientist wishing to embark on such work may find that differences in priorities and facilities, as well as in local regulations and pathology practice, require him or her to follow specific procedures. Nevertheless, an outline of a general approach to collection of brain material at autopsy is given below.

Once a patient has died and all appropriate consent has been obtained (i.e., permission for autopsy with explicit or implicit consent for removal of tissue for research), preparations can be made to obtain the brain. The body should be moved to refrigerated (4°C) storage, under these conditions the brain cools slowly, tak- ing some 15 h to cool to below 10°C (Spokes and Koch, 1978). At this point, two opposing factors need some consideration. First, there is the desire to obtain a neurochemical profile that best reflects the premortem state of the brain. This is offset by the fact that most biochemical changes occur in the first few hours after death as the tissue equilibrates with its anoxic state and before the temperature has dropped substantially. Thus, certain studies, e.g., assessment of metabolic activity, transmitter uptake and release, require fresh tissue taken only a few hours postmortem. However, it is apparent that many molecular investigations of human tissue are relatively tolerant of postmortem delay, whereas cause of death and agonal state are far more important factors.

Once the brain has been removed, it is normally weighed and possibly photographed, and the gross morphology is assessed. At this stage, the tissue needs to be prepared according to the even- tual use(s); this might involve freezing as a whole or as hemispheres prior to subsequent slicing and dissection of individual brain regions, freezing rapidly in thick slices prior to cryostat sec- tioning, or fixation in neutral buffered formalin or more specialized fixatives for histological or other cytological studies (see


Subheading 6.1.). Often, one hemisphere is chosen for specialized research study, whereas the other is employed for routine histological assessment. These sections, along with clmical data, may be used by the pathologist to confirm diagnosis and to provide an indication as to the possible presence of confounding factors, such as the neuronal changes of Alzheimer’s disease or other microscopic signs of cellular degeneration. Clearly, this procedure 1s not normally appropriate for research projects involving studies of laterality, as these require both hemispheres to be treated rden- tically.

Much “banked” tissue is stored frozen after dissection. Slow freezing may give rise to freezing artifacts (i.e., formation of mrcroscopically “large” ice crystals), and so is not always suitable for studies such as immunocytochemistry or zn situ hybridization histochemistry; however, regional immunohistochemical studies have been successfully undertaken using sections prepared from such slow-frozen samples. This tissue is typically used for neurochemical analysis of neurotransmitters, metabohtes, or enzymes, for studies involving radioligand receptor assays and radioimmunoassays, or as a source of DNA or RNA.

Alternatively, a brain or hemisphere may be cut into slices while still fresh (typically these slices will be anatomically defined and therefore constant between brains) and fast-frozen in a chamber containing either liquid nitrogen vapors or liquid-nitrogen-cooled isopentane. As freezing artifacts produced by this method are generally milder, these slices may then be dissected into blocks containing areas of interest, which can then be cut on a micro- tome in a cryostat to prepare slices for both histologrcal and neurochemical assessment, thm slices for receptor autoradiography, immunocytochemistry, or in situ hybrrdizatlon, and thicker slices for neurochemical assay using, for example, the grid or punch microdissection techniques. Snap freezing with liquid nitrogen is also the preferred method for storing biopsy specimins. Other pieces of tissue may be used in molecular biology studies. Obvi- ously, these procedures are intended to obtain the maximum amount of information from a tissue sample, and there may be times when tissue prepared in other ways may be required; for example, fresh tissue may be requested to provide thick vrbratome sections or for embedding in resin to allow very thin sections to be cut and prepared for electron microscopy.


4. Safety

Although dissection probably represents the most hazardous procedure in the handling of human postmortem tissue, all neuroscientists who come into contact with postmortem brain samples should be aware of the potential dangers. Therefore some consideration should be given to the topic of safety. Special precautions for the performance of the postmortem or for the handling of unfixed specimens (for example, for immunocytochemistry or autoradiography of frozen sections) are important where there is a suspicion of infectious disease. Current data suggest that in cases where infections are present, it is tuberculosis and hepatitis which represent the most important risks (Andrion and Pira, 1994). Accordingly, all personnel exposed to potential hazard should be immunized, for example, against hepatitis B. There is currently much interest in the pathology of infectious disorders involving the nervous system, including AIDS and spongiform encephalo- pathies, for example, Creutzfeldt-Jakob disease (CJD). Although transmission routes for this disease are not as yet fully understood, transmission of CJD (via prion proteins) is known to have occurred through contaminated biological products from postmortem tissues. As the prion is difficult to destroy, even being resistant to fixation in formalin, stringent sterilization precautions must be taken with all instruments that come into contact with postmortem material. Some tissues and body fluids, for example brain and CNS, from patients with CJD are highly infectious and must be contained or incinerated, although other bodily fluids are not considered infectious (Steelman, 1994). Transmission to health care and pathology workers is possible, and there are reports of CJD in pathologists and physicians (Gorman et al., 1992; Berger and David, 1993), and although these cases may be coincidental, all infected postmortem brain tissue should be treated with caution.

It is unfortunate, then, that there are no generally recommended safety regulations. However, Britain does have advisory guidelines for the handling of unfixed human brain that recognize the potential pathogenicity of all brain tissue. Thus, it is recommend that all procedures resulting in tissue disruption (i.e., mechanical slicing, homogenization), and are thus liable to produce an aero- sol containing brain tissue, should be carried out in a microbio- logical safety cabinet. This should be of an exhaust-protective, open-fronted design and housed in a room set aside for such pur-


poses. Other manipulations are considered less hazardous and can be done under laboratory conditions at which time more common precautions are observed.

As more research groups are becoming involved in postmortem brain investigations, such considerations are being increasingly ignored. It is clearly expensive (and often prohibitively so) to equip a laboratory to meet these recommendations. It would be unfortunate if, in the future, it were “brain bankers” who were the subjects used to confirm the hypothesis of an infectious vec- tor in one or other neurological disease of unknown etiology!

5. Variables Affecting Postmortem Neurochemistry

Table 1 lists some of the factors that are known to have effects on various neurochemical species measured in brain tissue.

5. 1. Age

Age and sex of the donor are perhaps the most obvious influences that are easiest to control for when attempting to match experimental groups. In particular, many changes with age have been documented and these include reductions in catecholamines and glutamate decarboxylase (GAD), which are specific to particular brain regions (Spokes, 1979). In the human striatal dopamine system, a reduction in the number of dopamine transporters with age has been reported (Volkow et al., 19941, as has an age- related decrease in the number of dopamine D2 receptors, although this was not observed in the frontal cortex (Wang et al., 1996).

Many of the reports of neurochemical changes associated with aging have emerged through the interest in dementia of the eld- erly. An early report from Winblad et al. (1982) has summarized the alterations in various monoamine-related parameters that occur with age; many, but not all, of these are more profoundly changed in the dementia of Alzheimer’s disease. It is, however, important to assess the influence of other factors, particularly agonal state and cause of death, which are likely to differ between younger and older patients. Since, for example, bronchopneumonia is more likely to be a cause of death in an older group, a potential influence of this and similar diseases on the measurement of, say, GAD, may masquerade as an apparent age effect unless it is specifically controlled for.

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Table 1

Variables Potentrally Affecting Neurochemical Parameters m Human Brain Tissue

Antemortem A@ Sex Agonal state Cause and manner of death l’sychratrrc and neurological history Termmal drug treatment Previous long-term drug treatment Time of day Time of year Point in menstrual cycle (younger females)

Postmortem Delay before refrlgeratron Delay before brain removal Drssectlon technique Storage conditions of frozen tissue Time m fixative -

5.2. Agonal State and Cause of Death

A decrease in GAD does, in fact, occur in brain tissue from patients who die after a protracted illness as opposed to previously healthy subjects who die suddenly (Spokes, 1979). Perry et al. (1982) have undertaken a study of this effect of agonal state on a wide range of biochemical species and find that GAD is the only activity directly associated with neurotransmitter function that shows a significant difference. However, there is a drop in tissue pH (specifically discussed in Subheading 6.3.) and several amino acids, notably tryptophan (increased by over 200%), exhibit substantial increases in concentration in the chronically ill group.

Other causes of death may be thought likely to affect the neurochemical status of the brain; encephalitis, bronchopneumonia, meningitis, and hepatic coma are a few obvious examples. Cer- tainly, the last of these has been found to have profound effects on brain amino acids, particularly tryptophan (Weiser et al., 1978), as well as on the concentrations of 5-hydroxytryptamine (5-HT) and its specific binding site (Riederer et al., 1981). These authors also report losses in brain levels of dopamine-stimulated adeny- late cyclase in noncomatose cases of liver cirrhosis.


Immunocytochemical studies have shown that pyruvate decarboxylase, a marker for glial cells, is markedly affected by agonal state, although a second glial marker, glutamine synthetase is not affected in this way (Butterworth, 1986), indicating that choice of antibody may be important in studies of human postmortem brain tissue.

5.3. Drug Treatmenf

A wide range of neuroleptics, stimulants, antidepressants, anxiolytics, antiparkinsonian drugs, and other psycho- and neu- reactive medications have been shown to have effects on neurochemical parameters in experimental animals (e g., Damask et al , 1996). Neuroleptic drugs may also be responsible for cellular alterations, for example, haloperidol has been seen to cause synaptic rearrangements in the medial prefrontal cortex of rats (Benes et al., 1985).

It is not always clear whether these effects are paralleled by changes in the human brain (for reasons that have been discussed), although there are indications that neuroleptic (Reynolds et al., 1981a,b; Mackay et al., 1980) and antiparkinsonian drugs (Birkmayer and Riederer, 1983), among others, can induce neurochemical changes in human bran-t tissue equivalent to those observed in animal studies.

It should be remembered that drugs active on the cardiovascular system may well also have CNS activity: particularly notable are the beta blockers, Noradrenaline (NA) or dopamine may be given in relatively large quantities shortly before the death of subjects with acute heart failure who might otherwise appear to be “good” controls. This can lead to massive (i.e., over lOfold) increases in the concentration of these transmitters m certain bram regions. Administration of opiates, common in chronically ill terminal cases, has been observed to have effects on 5-HT and dopamine systems (Bucht et al., 1981; Elwan and Soliman, 1995) in addition to their inevitable influence on the opiate peptides. Cytotoxic chemotherapy for cancer is also likely to have effects on neuronal function, although, as with most drug treatments, a systematic study using postmortem brain tissue is lacking.

5.4. Cyclic Fluctuations With Time

It is well established that the pineal gland exhibits a profound circadian rhythm in the content of melatonin and its synthesizing


enzymes, and human postmortem studies have closely related these concentratrons to the time of death (Smith et al., 1981). There are indications that other neuronal systems exhibit a dependence on time of day. Perry and Perry (1983) have reported that two enzymic markers of cholinergic transmission, as well as muscarinic receptor binding, exhibit circadian variations. Hypothalamic NA and its metabolites, as well as dopamine and 5-HT in this region, have also been identified (Carlsson et al., 1980) as show- ing a dependence on hour of death. These authors have reported seasonal fluctuations in 5-HT and dopamine in the hypothalamus, with less notable variations in other brain regions, Pineal function also exhibits a circannual rhythmicity (Smith et al., 1981) as do some indicators of dopamine activity (Karson et al., 1984), although, as the latter authors point out, potential postmortem changes dependent on ambient temperature may well introduce artificial seasonal effects.

The menstrual cycle is likely to evoke neurochemical indrca- tions of a central effect, certainly female sex hormones can, in animal experiments, induce changes in several transmitter systems including dopamine receptors (Hruska and Silbergeld, 1980). Therefore, apart from the menstrual cycle, the sex of the subject would be expected to have effects, and certainly several indicators of 5-HT and dopamine systems in the brain differ between men and women (Gottfries et al., 1981).

5.5. Postmortem Delays

As we have mentioned, the past reluctance of many biochem- ists and molecular biologists to use human tissue as a research tool is based mainly on the delay of many hours (and sometimes days) between death and tissue availability for experimentation. The initial assumption is that in this time so many proteolytic, oxidative, or other changes will have occurred that there is little resemblance to the neurochemical status before death. This view is to some extent understandable when one considers the work done in animal experiments to minimize such changes: For certain studies involving analyses of transmitters, some groups con- sider decapitation and immediate immersal of the brain in liquid nitrogen to be inadequate and recommend killing the animal by high-power microwave heating of the head that inactivates the relevant enzymic processes in much less than a second!


It is, however, wrong to dismiss work on human brain tissue for this reason. Many enzymes, neuropeptides, and neurotransmitters are surprisingly stable postmortem, particularly when the body is refrigerated shortly after death (Bowen et al. 1977). How can this stability be assessed? First, a large group of control material with varying postmortem delays can be used to assess variation with time, although this does not indicate what changes occurred during the initial hours after death. Gottfries et al. (1981) have identified changes in some amine transmitters and their metabolites as well as increases m tryptophan and tyrosine with increasing postmortem delay using this method. Second, postmortem and biopsy tissues could be compared, although the very limited availability of the latter, along with even greater limitations in the brain regions that could be studied, prevent this method from being of general use. Third, an animal model of the cooling conditions of the human brain between death and autopsy can be used. Spokes and Koch (1978) first used this method by monitor- ing brain temperature after death and subsequently constructing a cooling curve for mice killed by cervical dislocation (i.e., with- out breaking the skin). They observed that while the activities of GAD and choline acetyltransferase in the brains were little changed (GAD stabilizing at 80% after about 24 h), dopamme concentrations dropped by about 50% and most of this occurred during the first 4 h after death. On the other hand, postmortem delay may not be as critical as first thought. Neuropeptides have been shown to exhibit no losses over 72 h (Emson et al., 1981) and a wide range of enzymic or receptor activities are also reported to be stable for several hours postmortem (Hardy and Dodd, 1983). For example, Cortes et al. (1989) found no effects of postmortem delay on dopamine Dl receptor distribution, and studies employing in situ hybridization have shown that postmortem delay exerts only minor effects on mRNA expression (Perrett et al., 1992; Barton et al., 1993).

5. 6. Controlling for These Factors

The above discussion has mentioned many potential influences on neurochemical measurements, all of which would be difficult, if not impossible, to correct or take fully into account; hence, the importance of choosing, when necessary, appropriate control groups. Matching for age and postmortem delay is usually straightforward. Many of the other variables, such as sex, time of

Neurochemistry of Postmortem Bran 337

death, and so on, will automatically show no significant difference between the groups. However, when the study involves a group of neurological or psychiatric cases, several factors inevitably will differ. The unknown influence of the various contribu- tory effects associated with hospitalization or institutionalization, and the possible differences in cause of death that may reflect this, are particular examples. Thus, it is common for patients with chronic and progressive motor dysfunction as occurs in Parkinson’s and Huntington’s diseases to be bedridden in the latter stages of life, frequently dying from bronchopneumonia or respiration pneumonia. “Normal” control subjects, on the other hand, often comprise a group of sudden death cases, often from cardiovascular causes. A further “institutionalized” or disease control group may permit such influences to be controlled for, albeit only after introducing further confounding factors. Ideally, such antemortem factors need to be controlled for; as discussed above, agonal state can have profound effects on a variety of parameters. However, tissue pH and/or tryptophan concentration are two measurements that can be made to determine whether agonal state is appropriately controlled for (see Subheading 5.2.)- pH can be measured in brain either at death or later in tissue homogenates; it appears to be stable in frozen tissue.

Drug treatment is another problem and it is frequently impossible to differentiate between drug effects and the disease pro- cess. In addition to the chronic effects of drug treatment discussed above (Subheading 5.3.), the presence of residual drug in the tissue may interfere with the experimental assay. It has been shown that the assumption that washing of tissue sections may elimi- nate drug from tissue preparations is wrong, even after an extensive cycle of homogenization/centrifugation washes, at least for some neuroactive drugs (Owen et al., 1979). Such an assumption may lead to misinterpretation of receptor binding studies; for example, in the apparent deficit in muscarinic receptors in schizophrenics receiving the muscarinic antagonist benztropine (Dean et al., 1996; discussed by Reynolds, 1996).

In many sample groups the number of variable parameters will outweigh the number of samples, so it is perhaps advisable to approach such studies with a different mental attitude than if one were planning an equivalent animal experiment. First, one should remember that in general there would be no equivalent animal study for the investigation (parameter X in disease state Y in the


human). Second, the variability of the subject matter, which is such a limitation in one sense, may also provide far more information than the “ideal” situation in which the subjects within a group were all the same age, with a similar cause of death and so on. For example, it has been shown that choline acetyltransferase activity in cortical tissue increases with age of death in subjects with Alzheimer’s disease, but not in controls, an observation that has as much importance for the understanding of this disease as the difference in the enzyme activities between the groups (Rossor et al., 1981a). The fortuitous identification of such a correlation would be impossible in groups of subjects with less individual variability

6. Techniques and Applications

A wide range of neurochemical determinations can be applied to postmortem brain tissue, dissected as described above, using fairly standard biochemical techniques. These permit comparrsons of, for example, the neurochemistry of different regions of the brain or between the same region in different subject groups. The dlf- ferent freezing and dissection procedures now in common use (summarized in Table 2) also permit more precise study of the neurochemical anatomy of the human brain using a number of techniques, some of which will be discussed further.

6. I. Histochemistry and lmmunocytochemistry

The various histochemical techniques, in partrcular immunohistochemistry, are widely used in the neurosciences and are equally applicable to human tissue as to animals, except in those cases in which changes postmortem or during tissue preparation remove or inactivate the relevant marker. Histochemistry in general requires fixation of relatively fresh tissue so that cellular integrity is preserved. For some histochemical studies, for example staining for enzymes such as NADPHdiaphorase, paraffin sections are not acceptable, and fresh frozen or vibratome sections must be employed. Histochemistry, as well as being used to provide detailed anatomy of a transmitter or enzyme, has also been employed to define anatomical nuclei prior to “gross” neurochemical studies, for example, identifying the cholinergic cells of the substantia innominata and their changes in Alzheimer’s disease (Rossor et al., 1981b).

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Table 2

Tissue Preparations Applicable to Neurochemrcal Studies of Human Postmortem Brain

Tissue preparation Application

Fixation in neutral buffered formalin Histology or paraformaldehyde, blocks Immunocytochemistry embedded in paraffin In sttu hybridization

Conventional freezing of hemispheres Neurochemical analysis of transmitters, metabohtes, enzymes

Radioligand binding assays Radioimmunoassays Molecular genetics studies

immunocytochemistry and rn SW hybrrdization (when freezing artifacts are mild)

Fast freezing of tissue blocks or slices Immunocytochemrstry In situ hybridization Autoradrography Molecular genetrc studies

Slow freezing in isotonic sucrose Functional studies including mrtochondrial respiration and uptake activity studres

Peptide transmitters are fairly stable postmortem, and are particularly suitable subjects for immunohistochemistry. Studies of the distribution of particular neuropeptides in neurological and psychiatric disorders have provided some interesting data (e.g., Hunt et al., 1982; Ang and Shul, 1995). Although an ever-increasing number of antibodies suitable for use in postmortem brain research are commercially available, the limiting factor in studies of this type is the fact that well-prepared tissue is not commonly available. Immunocytochemical staining may be affected both by the type of fixative used and the length of time the tissue is stored in fixative. Archival material, typically fixed for long periods in formalin is usually easier to obtain, but is only suitable for studying certain antigens, for example, peptides such as vasopressin, VIP, and NPY which are resistant to such prolonged fixation times (e.g., Swaab, 1982; Uylings and Delalle, 1997). Frozen cryostat or vibratome sections may be preferable to paraffin sections for immunochemical studies, as freezing does not typically cause a


loss of antigens, this being especially important when studying antigens that are easily masked. Having said this, an increasing number of techniques that allow antigen retrieval in archival material are available. For example pretreatment with formic acid is widely recognized as improving staining of PA4 (Kitamoto et al., 1987) and proteolytic enzyme digestion, especially by trypsin, is widely used to increase antigen retrieval for some antibodies (Battiflora and Kopinski 1986). Finally, the more recent technique of microwaving, first described by Shi et al. (19911, is known to improve significantly staining for certain antigens (Fig. 1). The masking of antigens is typically most pronounced in tissue fixed with neutral buffered formalin, and although when other fixatives, for example, Bouin’s fixative, are used, this is less of a problem, these often have disadvantages and are accordingly not widely used. For studies involving electron microscopy gluteraldehyde fixation is recommended, prior to embedding in resin. Recently, it has been described that variations of formalin, including zinc or calcium formalin (reviewed by Dapson, 1991) are good for pre- serving antigens, and it may be possible that in the future these will become more widely used.

6.2. Receptor Autoradiography

Autoradiography can be used as a visualization technique for immunohistochemical studies by employing radiolabeled antibodies; however, the availability of high specific activity radioligands for receptor studies has been followed by the application of autoradiography to the identification of neurotransmitter receptors m sections of brain tissue. Research groups are increasingly investi- gating human brain in this way, using thin sections prepared from frozen tissue (e.g., Palacios et al., 1980; Lahti et al., 1995). Because receptors are, on the whole, very stable in the postmortem period, the technique as developed in, and widely applied to, animal tissue can be used generally with human samples. The same advantages and disadvantages over “test-tube” receptor binding apply, these include the generally lower amounts of nonspecific binding and the greater difficulties in kinetic characterization of the receptor. One obvious advantage is the greater level of resolution that receptor autoradiography can offer; this may permit assessment of the laminar distribution of a receptor site in the neocor- tex. However, the lack of opportunity to undertake extensive washing of tissue, as can be done u-r the preparation of homo-


Fig. 1. Parvalbumin stained neurons in paraffin sections of human frontal cortex following microwave treatment.

genates for receptor binding, introduces major limitations. Radioligands with relatively low (i.e., micromolar, as opposed to nanomolar) receptor affinities are difficult to use, since a brief wash of the tissue to remove nonspecifically bound ligand may also significantly disturb the equilibium and result in losses of specific binding. Similarly, competitive binding at sites that recognize an endogenous ligand/transmitter, such as glutamate, that is normally in high concentration in the tissue and not readily removed


by superficial washing, may also result in inadequate radioligand binding. Careful choice of the ligand/site may allow the researcher to avoid some of these pitfalls; for example, NMDA receptors may be determined by autoradiography using radiolabeled MK-801, which binds noncompetitively to the ion channel site, rather than with compounds binding competitively to the glutamate binding site.

Finally, the problems associated with residual therapeutic drugs, mentioned in Subheadings 5.3. and 5.6., are inevitably greater in autoradiographic studies than with homogenate binding.

6.3. mRNA Studies and In Situ Hybridization

The growth in molecular biology has made a huge impact on neurology, one example being the identification of a gene containing an increased number of trinuceotide repeats in Huntington’s disease. The application of modern molecular techniques to human postmortem brain tissue started with the isolation of active mRNA (Gilbert et al., 1981), and was found to lend itself well to the investigation of hereditary neurological disorders or chromosomal abnormalities such as Down’s syndrome (Whatley et al., 1984). Per- haps the most widely used approach to determining nucleic acid function in brain tissue is in situ hybridization histochemistry, and an example is shown in Fig. 2.

The practical application of this technique to human brain tissue has been discussed in detail previously (e.g., Heath et al., 1996) and its specific application to the study of human neurological and psychiatric diseases has also been considered elsewhere (Harrison and Pearson, 1990). In situ hybridization studies are generally undertaken using freshly prepared frozen cryostat sections, postfixed with paraformaldehyde, although, as mentioned previously, tissue prepared in this way may not always be available, and in these case, paraffin sections may be used. Where paraffin sections are used, proteolytic digestion by, for example, Type XXIV protease, must be employed. It should also be noted that the fixative used may have an effect; glutaraldehyde fixation commonly causes a diminished signal when using this technique. Although paraffin sections can be used for studies of mRNA expression, results tend to be variable, especially when radiolabeled oligodeoxyribonucleotide probes are used, and therefore, the use of frozen sections is preferable for quantitative studies. If there is no alternative to using paraffin sections, results may be improved by using nonradioactively labeled full-length RNA probes (Heath


Fig. 2. API? mRNA expression in section of human hippocampal formation. (Courtesy of Prof. R. C. A. Pearson.)

et al., 1996). There appears to be no evidence of any deleterious effects caused by long term storage of frozen blocks or pretreated sections, although freeze-thaw cycles should be avoided.

The majority of evidence suggests that postmortem delay exerts a relatively minor effect on recovery of mRNA (reviewed by Barton et al 1993). Occasionally, there may be an effect on individual mRNA’s in specific brain areas, especially when the postmortem delay is over 48 h, and, therefore, this variable cannot be completely ignored. On the other hand, agonal state appears to exert a good deal of influence on mRNA expression, for example coma


(Harrison et al., 1991), hypoxia (Burke et al., 1991), and pyrexia (Harrison et al., 1994) have all been known to affect message. As pH has been found to be a good indicator of agonal state, pH decreases with increasing age and with agonal state severity, but is not related to PM delay or hypoxic histological changes (Harrison et al., 1995), it may be more valuable to use pH as a marker, rather that relying on retrospective measures.

6.4. Microdissection

The dissection method generally applied to “brain banking” (see Subheading 3.) can hardly be described as microdissection, involving, as it does, fairly crude dissection of structures from brain sections several millimeters thick. However, the reader would be reminded that an animal brain of a gram or so in weight would certainly require microscopic techniques to obtain an equivalent accuracy. Nevertheless, even greater anatomical detail can be obtained with classical biochemical assays after using microdissection methods. The use of these methods in the study of animal tissues is described by Palkovits (19851, who pioneered punch microdissection. They are in many ways even better suited to sub- regional mapping of the human brain with its more complex anatomy yet larger volume of most regions and nuclei. The application of the “punch“ and “grid” microdissection techniques to frozen sections of human postmortem brain tissue has been reviewed (Aquilonius et al., 1983; Aquilonius, 1986). Either small blocks of tissue (e.g., striatum or a crosssection slice of spinal cord) or, with the use of an appropriate large-section cryomicrotome, a whole brain or hemisphere can be used to provide frozen sections of tissue from which punches can be taken or a grid of tissue pieces prepared for biochemical analysis. These techniques have been used to study the heterogeneity of various biochemical species within regions of the brain. One interesting study was that of Oke et al. (1978), who, by mapping the distribution of NA within the human thalamus, identified a pattern of lateral asymmetry for this transmitter.

6.5. Functional Studies

So far the discussion has been restricted to static parameters in postmortem tissue: the measurement of receptor densities, transmitter concentrations, antigen distributions, and enzyme activi-


ties. Few realize, however, that a range of functions normally associated with extremely fresh tissue can also be measured in preparations of brain many hours postmortem. The initial indication that some dynamic cellular processes remain is that synaptosomes prepared conventionally from frozen tissue exhibit respiratory activity (Hardy et al., 1982). Thus, there is a support- ing mechanism for fueling those processes, such as uptake and release of transmitters, which require energy. Uptake of catecholamines, 5-HT, and transmitter amino acids has indeed been observed by several groups, as have active depolarization-sen- sitive release processes (reviewed by Hardy and Dodd, 1983). These workers also describe an even more surprising phenome- non: the ability to store tissue deep-frozen for long periods with- out substantial losses of synaptosomal viability. They use a procedure of slow freezing followed by rapid thawing to obtain high yields of functioning synaptosomes (Hardy et al., 1983). Bowen et al. (1982) have shown that metabolic activity and ace- tylcholine release occur in cortical tissue prisms taken shortly after death. This group has also been able to preserve such tissue in a viable form by the use of dimethyl sulfoxide to protect against damage from freezing and thawing (Haan and Bowen, 1981). The methodologies used to study functional transmitter neurochemistry in postmortem human brain are discussed further by Dodd et al. (1988).

The use of these procedures to assess neurotransmitter uptake and release is clearly underused. Their application has, if any- thing, decreased over the past decade as more straightforward preparation procedures compete for the limited availability of autopsy tissue. Certainly, there was no mention of this approach in a recent handbook on “brain-banking” (Cruz-Sanchez and Tolosa, 1993). Further work is required, particularly in their application to disease states, before one can assess the value of these functional measurements as a research tool. The substantial variation in absolute functional activity (V,,,) between individual samples due, for example, to agonal state and postmortem delay, will probably preclude simple comparisons, although the kinetic parameters (KM) should be more stable and may well permit the identification of functional abnormalities in neuropsychiatric disease. These preparations can be stored to retain viability almost indefinitely with potential for future use when neuroscience methods have even more to offer.


7. Neuropsychiatric Disease

The major motivation to work with postmortem brain tissue is to contribute to our understanding of the neurological and psychiatric diseases. This has been done with great success in Parkinson’s disease. Huntington’s disease has also attracted a lot of scientific interest, although probably the greatest effort in terms of defining a molecular pathology and in searching for clues indicative of pathogenic mechanisms has been put into the study of Alzheimer’s disease. Yet despite all the research and biochemical data that have emerged over the past two decades, no therapeutic breakthrough comparable to that for Parkinson’s disease has been made. Can we identify why this is so?

Several other transmitter abnormalities have been reported in the Parkinsonian brain, but the sole major dysfunction appears to be in the dopaminergic nigro-striatal tract as indicated by a loss of striatal dopamine typically over 80%. It is this deficit, which underlies the motoric symptoms of Parkinson’s disease, and which is directly addressed by L-dopa medication. Only relatively recently have the (less profound) losses in cholinergic, sero- toninergic, and noradrenergic systems been considered as important, potentially underlying the dementia and depressive symptoms that often occur in Parkinson’s disease.

Unfortunately, neurotransmitter deficits are often multiple and more generalized in other neurological diseases. The well- established loss of cholinergic innervation of the cortex in Alzheimer’s disease has led to the investigation and trial of a variety of cholinergic therapies, of which the recently introduced cholinesterase inhibitor, donepezil, has demonstrated a useful clinical efficacy. This therapeutic effect is, however, not consistent in all subjects, reflecting perhaps the variable contribution that is made by the cholinergic system to the total sum of neuronal deficits underlying the symptoms of the disease. Thus, there are also substantial losses in the NA- and 5-HT-containing mesocortical pathways (Winblad et al., 1982), although biopsy studies suggest that glutamatergic deficits are an important com- ponent to the overall pathology (Bowen et al., 1989).

Postmortem neurochemistry and histology have been applied to a range of other disorders not classically considered to be pri- marily neurological or psychiatry. Thus sudden infant death syndrome has been usefully investigated in this way (Lucena and


Cruz-Sanchez, 19931, with findings that include gliosis and cer- ebellar abnormalities, while histological and neurochemical pathologies of the brain in AIDS have revealed many neuronal and neurotransmitter deficits, One example of these findings is the Parkinson-like decrement in striatal dopamine that occurs in many cases (Sardar et al., 1996) and, in addition to providing an understanding of the motor dysfunction and neuroleptic sensitivity reported in AIDS patients, indicates the potential value of dopaminergic therapy in such cases.

The problem with the biochemistry of the psychiatric diseases is quite the opposite: a paucity of confirmed reports of neurochemical changes. Nevertheless, the observations of consistent, if subtle, neuroanatomical and neurohistological differences in schizophrenia has encouraged a renewal of interest in postmortem studies of the brain in this disease. Concordance in one biochemical find- ing is apparent: the density of dopamine D2-like receptors is increased. It is over the interpretation of this observation that there has been much dispute, although the weight of the evidence, from results in untreated humans and from animal studies, would appear to indicate that the change in receptor number is a response to antipsychotic medication and not an effect of the disease itself (Reynolds, 1989) This conclusion is supported by almost all studies of dopamine receptor density in vivo in unmedicated schizo- phrenic patients using positron emission tomography (PET) or its cheaper cousin, single photon emission tomography WET). These imaging techniques are now addressing, in living patients, many of the questions that were previously only answerable by human postmortem studies, with all the inherent limitations of this latter technique that have been described in this chapter. In addition, PET, SPET, and the imaging (MRI) and spectroscopic (MRS) techniques developed from nuclear magnetic resonance, are being used to investigate a variety of parameters not accessible in autopsy tissue. Thus, in vivo dynamic processes can be monitored; appropriate choice of radioligand in PET can permit the determination of relative levels of neurotransmitter release, whereas MRS has been used to identify differences in phospholipid metabolism, for example.

These new imaging techniques offer much at present, and even more for the future, to excite the interest of the neuroscientist com- mitted to understanding biochemical, physiological, and pharmacological aspects of brain disorders. However, they do not


completely supersede or replace the postmortem study. Results from imaging and MRS studies are often complex and have required new computational and statistical approaches for their analysis; there are many components to the kinetics and dynamics of, say, a radioligand introduced into the blood and being monitored in the brain. These components cannot readily be differentiated; there may well be a role in the future for complementary kinetic studies on postmortem tissue. More important, postmortem tissue can be used to follow up, confirm, or otherwise, elaborate on the neurochemical clues provided by these high technology approaches.

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10 neurochemistry of human postmortem brain

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