Neurochemical Research, Vol. 6, No. 7, 1981

Comment

POSTMORTEM STABILITY OF SOMATOSTAT IN-LIKE IMMUNOREACTIVITY

IN MOUSE BRAIN UNDER CONDITIONS SIMULATING HANDLING OF HUMAN

AUTOPSY MATERIAL

P E T E R D A V I E S A N D A U R O R A T H O M P S O N Department of Pathology

Albert Einstein College of Medicine 1300 Morris Park Avenue Bronx, New York, 10461

Accepted March 16, 1981

The postmortem stability of somatostatin-like immunoreactivity (SLI) has been investigated in mice cooled after death at a rate which approximates the rate of cooling of the human brain in situ under normal mortuary conditions. Significant changes in SLI levels were observed within the first few hours after death, but concentrations measured 6-24 hr after death seem to be similar to those existing at the time of death.

INTRODUCTION

Somatostatin (growth hormone release-inhibiting hormone or factor) was originally isolated and characterized by Guillemin and associates from bovine hypothalamus (1-3). Recent studies have shown that in the rat, less than 25% of the total brain somatostatin is contained in the hypo- thalamus (4), the remainder being found in regions such as the cerebral cortex, amygdala, septum, preoptic area and various midbrain and brain stem nuclei (5, 6). Several studies have suggested that this extrahypoth- alamic somatostatin might function as an inhibitory neurotransmitter in the mammalian brain (7-9).

Although there have been a few studies of somatostatin in human brain (10, 11), we have been concerned about the possibility that concentrations

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788 DAVIES AND THOMPSON

in brain tissues may alter in the period between death and removal of samples at autopsy. We have investigated this question using the protocol reported by Spokes and Koch (12). In these studies intact mouse carcasses are allowed to cool in an incubator at a rate which approximates that at which the human brain cools in situ under normal postmortem conditions. Assay of mouse brain tissues removed at various intervals after death allows estimation of the rate of postmortem autolysis of the substance under study.

EXPERIMENTAL PROCEDURE

In our investigations, 52 male BALB/c mice were killed by cervical dislocation and 44 of them were placed in an incubator at 37~ The brains of the remaining 8 mice were rapidly removed, and samples of frontal cortex were frozen on dry ice for subsequent assay of somatostatin. The temperature of the incubator was allowed to fall at the rate shown in Figure 1, this approximating the postmortem rate of cooling of the human brain in situ, as determined by Spokes and Koch (12). The core temperature of the mice follows closely the temperature of the incubator. Mouse carcasses (4-8) were removed from the incubator at time periods indicated, and frontal cortex samples were removed and rapidly frozen.

Tissue samples were placed in boiling 2 M acetic acid for 10 min, and then homogenized, Aliquots of homogenates were taken for protein determination (13), and the remaining

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FIG. 1. Postmortem changes in the concentration of somatostatin-like immunoreactivity in mouse frontal cortex are shown by the solid circles, which are mean values of determinations. The number of samples is shown for each point, and the bars represent the standard errors of the mean. Asterisks are used to indicate values which are significantly different from the zero-time concentration (P less than 0.05, Student 's t test). The insert shows the rate of cooling of the incubator.

POSTMORTEM STABILITY OF SOMATOSTATIN 789

portions were centrifuged at 15,000 g for 30 min. Portions of supernatants were freeze-dried, and the residues were redissolved in phosphate-buffered saline, pH 8.2 containing 0.1% bovine serum albumin. Recovery of [~zsI]-Tyrl-somatostatin or synthetic somatostatin added to tissue samples was 89-93%. S0matostatin-like immunoreactivity (SLI) was measured by radioimmunoassay, using a rabbit antiserum raised against a somatostatin-bovine serum albumin complex prepared as described by Arimura et al. (14). The assay buffer was phos- phate-buffered saline, pH 8.2, containing 0.1% BSA. The serum was used at a final dilution of 1:48,000, with [~zsI]-TyrLsomatostatin (12001~Ci/p.g, New England Nuclear Corp., Bos- ton, Massachusetts) as tracer. Bound and free tracer were separated with dextran-coated charcoal after incubations of 48 hr. Binding of tracer with no added somatostatin was 38%, and the minimum detectable concentration of synthetic somatostatin was about 5 pg/tube. Assay of serial dilutions of mouse brain extracts produced displacement curves with slopes identical to those obtained with synthetic somatostatin: Logit plots of displacements vs. concentration of synthetic somatostatin were linear over the range of 8-512 pg somatostatin. The presence of ! ixg/tube of glucagon, insulin, proinsulin, gastrin, neurotensin, substance P, thyrotropin-releasing hormone, or cholecystokinin octapeptide did not displace labeled somatostatin from the antibody.

RESULTS AND DISCUSSION

Concentrations of somatostatin-like immunoreactivity in mouse frontal cortex at various times postmortem are shown in Figure 1. As indicated in the figure, mean concentrations were significantly reduced at 30 rain, 1 hr, 2 hr, and 4 hr postmortem, as compared to the zero time point. However, at 6 hr postmortem, the mean concentration of somatostatin- like immunoreactivity was not significantly different from that at time zero and was significantly elevated when compared to those at 30 rain and 1 hr postmortem. At 8, 18, and 24 hr postmortem, the concentrations were not significantly different from those at zero time.

Our data seem to indicate that measurements of concentrations of SLI at times 6-24 hr after death would provide reasonable estimates of levels in tissue from freshly killed animals. If these results can be extrapolated to studies on human autopsy specimens, the values obtained also ought to reflect those present at the time of death, provided at least 6 hr elapse before the brain is removed. While there are undoubtably differences between mouse and human brain, this model system for the study of postmortem stability seems to be the best available at this time. Other brain peptides such as substance P (15) and vasoactive intestinal peptide (16) also seem to be stable in similar model systems. Our preliminary results of studies of cerebral cortex from 24 human brains have so far failed to reveal correlations between concentrations of SLI and post- mortem time.

Explanation of the changes in SLI concentration which occur within the first few hours of death is difficult at this time, and further studies are

790 DAVIES AND THOMPSON

clearly required. High-molecular-weight putative somatostatin precursors have been identified in some tissues, (17) and investigation of the possible differential sensitivities of these molecular species to postmortem deg- radation might be informative.

ACKNOWLEDGMENTS

This work was supported in part by NIH grants AG-01066, NS-02255, and NS-03356. We thank Dr. Robert D. Terry and Dr. Robert Katzman for advice and support in this work.

REFERENCES

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POSTMORTEM STABILITY OF SOMATOSTATIN 791

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