Metabolically Active Synaptosomes Can Be Prepared from Frozen Rat and Human Brain

Abstract: Nerve ending particles (synaptosomes) were prepared from pieces of rat and human brain and from brain homogenate that had been frozen and thawed under a variety of conditions. Their purity, as judged by electron microscopy, and performance in terms of a number of metabolic and functional parameters [accumulation of tissue potassium, respiration, release of transmitter amino acids, and the responses on these indices to depolarisation by veratrine (VX)] were compared with those of fresh tissue-derived synaptosomes. It was found that rapid freezing and/or slow thawing severely impaired the subsequent performance of incubated synaptosomes. In contrast, synaptosomes from tissue frozen slowly and thawed rapidly showed relatively good retention of morphology and metabolic performance. It was better to use whole (1-5 g) pieces of tissue than tissue homogenate: the synaptosome fraction from frozen tissue pieces contained 80% of the proportion of identified synaptosomes found in the fresh tissue synaptosome fraction, its respiratory rate was 65%, and its tissue potassium content 70% of that of fresh controls. Moreover, it responded to VX or potassium stimulation by showing increased respiratory rate, decreased tissue potassium, and increased release of neurotransmitter amino acids, to an extent that was comparable to that of fresh tissue fractions. Thus, preparations from frozen rat and human brain were shown to be metabolically and functionally active, and can be used for a variety of neurotransmitter-related studies.     

A cell-free approach to the study of membrane trafficking in frozen rat brains potentially applicable to frozen autopsy material

Summary A cell-free transfer system was used to measure capacity of brain membranes to support membrane renewal. To study transfer in brain, radiolabeled donor microsome fractions were prepared using brain slices from rats or frozen human brain autopsy specimens. Acceptor fractions, prepared from fresh or frozen rat brain or frozen human brain autopsy specimens, were immobilized on nitrocellulose. The complete reconstituted transfer system contained ATP plus ATP-regenerating system (or NADH) as a source of energy and brain cytosol. Slices of frozen brain incorporated acetate into membrane lipids with approximately the same efficiency as fresh brains. This efficiency declined with storage at 4 °C but only slowly. Donor fractions labeled with acetate from frozen slices exhibited specific transfer (37 °C minus 4 °C) of labeled membrane lipids with efficiencies comparable to fresh. The acceptor fraction could be prepared either from fresh or frozen material. Transfer was on the average two-fold stimulated by ATP at 37 °C compared to no ATP. Transfer also was stimulated by NADH. Analysis of linear transfer rates between 0 and 30 min revealed no significant effect of delay time or of time of prolonged storage on transfer efficiency beyond an initial decline of ca. 25% observed within the first two weeks after freezing. A decline of transfer was obtained with brains as the animals aged.     

Effects of tissue storage and freezing on brain glutamate uptake

Glutamate uptake appears to be stable when measured in rat striatal synaptosomes from tissue stored for up to four hours post-mortem at 25°C. Between four and eight hours storage at room temperature there is a sharp 70% decrease in uptake. Freezing of tissue on dry ice, storage at 4°C for up to 7 days and at ?80°C for 5 days results in 20–30% residual glutamate uptake. Quantitatively similar data can be obtained in eight extrastriatal brain areas. Kinetic analysis of glutamate uptake in stored and frozen tissue reveals the loss of the majority of both sodium-dependent high affinity and temperature-sensitive low affinity sites (v_max-values) while the respective K_m-values are not significantly changed. Pharmacological properties of the high affinity uptake versus a number of specific and metabolic uptake inhibitors remain unaltered by the storage and freezing procedure. The tissue treatment chosen for the present study roughly corresponds with the preparation of human post-mortem brain tissue for enzyme-, receptor-binding- or neuro-transmitter assays. It therefore seems conceivable that meaningful uptake studies can be performed on human autopsy material, thus adding an important parameter to the battery of neurochemical markers already accessible for post-mortem $\text{in}$$\text{vitro}$ examination.     

Characterization of Postmortem Human Brain Proteins by Two-Dimensional Gel Electrophoresis

Abstract: The proteins of membrane and cytosol fractions from frozen human postmortem brain were analyzed by two-dimensional gel electrophoresis (isoelectric range: 5.1–6.0) and both Coomassie-blue and ammoniacal silver staining. Cytosol preparations were analyzed from six different postmortem brains from patients with various neurologic diagnoses and immediate causes of death. Intervals between death and brain freezing (−70^oC) ranged from 2 to 20 h. The vast majority of proteins detected in these cytosol fractions had identical molecular weights and isoelectric points in each of six human brains examined. However, in some tissue samples tubulin was either quantitatively decreased or undetectable. The possibility that this partial or complete depletion of tubulin was related to postmortem interval and/or brain freezing was studied using rat forebrain tissue. Rat brain incubated at room temperature for up to 24 h did not reproduce the changes seen in the region of human cytosol tubulin. However, other changes seen in the two-dimensional electrophoretic pattern of rat cytosol proteins did relate to postmortem interval, brain freezing, or both. Rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum were prepared from three human brains, with highly reproducible two-dimensional patterns. Protein analysis of these membrane fractions revealed that human RER contained significant amounts of tubulin, in contrast to rat RER which contained no detectable tubulin. This discrepancy was elucidated by allowing rat brains to remain at room temperature for 24 h before freezing; gels of rat RER prepared from this tissue showed that tubulin subunits were present.     

Protection of Neocortical Tissue Prisms from Freeze-Thaw Injury by Dimethyl Sulphoxide

Abstract: Neocortical tissue prisms prepared from rat and human brain were frozen to -196°C by a two-step freezing procedure and 10% dimethyl sulphoxide as cryoprotectant. Frozen and thawed rat neocortical prisms incorporated glucose into acetylcholine and carbon dioxide at 89% and 86% of control values, respectively, and noradrenaline uptake into frozen and thawed rat prisms was 94% of the control value. Frozen and thawed prisms from three human neocortical specimens showed a similar degree of protection from freeze-thaw injury.     

Glial fibrillary acidic protein from bovine and rat brain. Degradation in tissues and homogenates.

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol wt. polypeptide in bovine tissues incubated at 24 degrees C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000-40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 degrees C. At pH 8.0 PROTEOLYSIS OF The glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Schistosoma mansoni: a comparative study of artificially transformed schistosomula and schistosomula recovered after cercarial penetration of isolated skin.

A comparison was made of the ultrastructure, development and antigenic nature of the surfaces and of the viability of three types of Schistosoma mansoni: schistosomula formed after cercariae had penetrated isolated skin (SS) schistosomula produced after mechanical separation of cercarial tails from bodies (MS), and schistosomula transformed from cercariae after incubation in fresh rat serum (RS). Within 2h of transformation, the surface membranes of all three types of schistosomula had changed from trilaminate to heptalaminate structures and SS and MS had lost their cercarial glycocalyx. Initially a dense amorphous material was demonstrated on the surfaces of RS, which was thought to be the result of an interaction between a factor in rat serum and the glycocalyx; this material was greatly reduced within 2 h of transformation. The pre-acetabular glands of SS were emptied while those of MS and RS retained their contents. Immunofluorescent studies showed that all schistosomula bound serum from mice immune to S. mansoni, but the binding was stronger with MS and RS. The mixed agglutination reaction demonstrated the presence of human A and B blood group-like antigenic determinants on approximately 30% of 3h old SS; these determinants were not detected on MS or RS. In vitro, the development of MS and RS was similar to SS; the first schistosomula reached the "gut-closed" stage by day 10; 50-70% of SS reached this stage by day 12, in contrast to only 25-50% of MS and RS. Between 28 and 45% of all schistosomula developed to maturity when injected intravenously into mice. It was concluded that the two types of artificially prepared schistosomula fulfil the main criteria of transformation from cercaria to schistosomulum. Further, it is suggested that MS are the most appropriate source of material for immunochemical and physiological studies.     

Differential expression of BK channel isoforms and β-subunits in rat neuro-vascular tissues

We investigated the expression of splice variants and β-subunits of the BK channel (big conductance Ca²⁺-activated K⁺ channel, Slo1, MaxiK, K_Ca1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An α-subunit splice variant X1_+ 24 was found expressed (RT-PCR) in nervous tissue only where also the SS4_+ 81 variant was dominating with little expression of the short form SS4₀. SS4_+ 81 was present in some cerebral vessels too. The SS2_+ 174 variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported the finding of SS4_+ 81 and SS2_+ 174 in vascular smooth muscle and trigeminal ganglion. β-subunits β2 and β4 showed high expression in brain and trigeminal ganglion and some in cerebral vessels while β1 showed highest expression in blood vessels. β3 was found only in testis and possibly brain. A novel splice variant X2_+ 92 was found, which generates a stop codon in the intracellular C-terminal part of the protein. This variant appears non-functional as a homomer but may modulate the function of other splice-variants when expressed in Xenopus oocytes. In conclusion a great number of splice variant and β-subunit combinations likely exist, being differentially expressed among nervous and vascular tissues.     

Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use

A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at −70°C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence of viable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities.     

Variability of laminin immunoreactivity in human autopsy brain

Summary Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

The apparent target size of rat brain benzodiazepine receptor, acetylcholinesterase, and pyruvate kinase is highly influenced by experimental conditions

Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme.     

Variability of laminin immunoreactivity in human autopsy brain.

Laminin immunoreactivity is thought to be masked in formalin-fixed sections since proteolytic treatment is required to unmask it. We analyzed this masking with frozen and formalin-fixed human autopsy brains obtained at various postmortem periods. In unfixed, frozen sections, intense immunoreactivity was invariably detected in vascular walls of entire sections. When such sections were postfixed in formalin, immunoreactivity was not diminished even after prolonged fixation. In vibratome sections of brain fixed in formalin in situ, immunoreactivity varied with postmortem delay: in most cases, immunoreactivity was weak and restricted to superficial cortical layers. However, the extent of immunoreactivity increased with postmortem delay. Two cases fixed after prolonged postmortem periods revealed moderate immunoreactivity throughout the sections. We also investigated rat brains processed without postmortem delay. In unfixed frozen sections, immunoreactivity again was observed throughout the sections, independent of the length of any postfixation. In vibratome sections of fixed rat brain, immunoreactivity was restricted to the cutting margins of the brain blocks and around a trauma-induced cortical lesion, regardless of how long the blocks had been kept in fixative. Our data suggest that postmortem proteolysis accomplishes similar unmasking of laminin antigen as digestion on paraffin sections and that such unmasking can also be effected by proteolysis induced by damaging tissue during cryostat sectioning of fresh tissue.     

The low affinity binding site for the cocaine analog, WIN 35,428 is an artifact of freezing caudate tissue.

Binding of the cocaine analog [3H] WIN 35,428 was investigated in rat and rabbit caudate. In membranes prepared from fresh tissue, [3H] WIN 35,428 binding was characterized by a single high affinity site with a Kd of 2.5 nM for the rabbit and 5.3 nM for the rat. In contrast, [3H] WIN 35,428 binding to membranes prepared from frozen tissue (stored at -70 degrees C) revealed two binding sites, a high affinity site similar to the one observed in membranes from fresh tissue and a low affinity site with a Kd of 39 nM for the rabbit and 65 nM for the rat. The low affinity WIN 35,428 binding site was observed only in membranes derived from frozen tissue, suggesting that it was an artifact produced by the freezing/thawing process.     

Effects of cysteamine on pro-somatostatin related peptides

Intracerebroventricular (icv) injection of cysteamine to rats produced a marked depletion of somatostatin-14-like immunoreactivity (LI) in all rat brain regions examined. The somatostatin-28 (SS28)-LI and SS28(1-12)-LI were generally not altered by the cysteamine treatment. Following subcutaneous injection of the drug similar depletions of hypothalamic SS14-LI was observed with no change in SS28-LI nor SS28(1-12)-LI. In vitro cysteamine significantly increased the basal release of SS14-LI and markedly potentiated the evoked release of SS14-LI from hypothalamic slices. At 10 mM cysteamine, enhanced release of SS14-LI from hypothalamic slices was still observed despite a marked depletion of tissue content of SS14-LI.     

Metabolic and functional studies on post-mortem human brain

The evidence that samples of human brain tissue obtained at autopsy may be used as starting material for the isolation of cellular and subcellular preparations which exhibit metabolic and functional activity when incubated in vitro has been reviewed. Supporting evidence has been found in data from model experiments which used animal brain as the source material. Active preparations have been obtained after considerable (up to 24 h) post mortem delays. Such findings are less surprising when the post mortem stability of key tissue components (enzymes, receptors, nucleic acids) and the retention of cellular integrity are examined. The data from these fields have been reviewed and their relevance to functional studies assessed. Studies which use human autopsy material must consider many additional sources of variation not found in experiments with animal brain and the major problems are briefly discussed. It is argued that functional experiments present few, if any, difficulties not already inherent in static analyses of autopsy material and some procedures which help to minimise these difficulties are outlined. Experimentation in this area is greatly aided by the finding that metabolically and functionally active preparations may be obtained from frozen tissue pieces. Dynamic studies provide a new approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of neuroactive drugs in man.     

An antiserum to the C-terminus of the Alzheimer amyloid precursor recognizes a soluble 70 kDa protein

A rabbit antiserum to the C-terminus of the putative brain amyloid precursor was used to probe Western blots of tissue proteins separated by SDS-PAGE. The antiserum specifically labelled a protein of approx. 70 kDa in the Tris buffer-soluble fraction of brain samples from rat, Alzheimer subjects, cases of young and old Down's syndrome, and age-matched controls. The 70 kDa protein was present in low concentrations in human liver and kidney, and was undetectable in human skeletal muscle. The 70 kDa protein may be a metabolite of the amyloid precursor.     

[3H]GBR-12935 Binding to the Dopamine Transporter Is Decreased in the Caudate Nucleus in Parkinson''s Disease

The specific binding of [3H]GBR-12935 to membranes prepared from human caudate nucleus is saturable (Bmax 1.36 +/- 0.18 pmol/mg protein), sodium dependent and of high affinity (KD 2.34 +/- 0.18 nM). Freezing of tissue from rat brain, or refrigeration followed by freezing, results in a small but significant (less than or equal to 20%) decrease in specific [3H]GBR-12935 binding when compared to the binding observed in fresh (nonfrozen) tissue, and this decrease may account, in part, for the differences in specific binding between rat and human brain membranes. Despite small differences in binding site density between fresh and frozen tissue there is a good correlation (r = 0.98; p less than 0.01) between the potencies of a series of drugs in displacing specific [3H]GBR-12935 binding to human caudate membranes and rat striatum as well as in inhibiting dopamine uptake in rat striatal synaptosomes (r = 0.96; p less than 0.01). The specific binding of [3H]GBR-12935 to membranes prepared from the caudate nuclei of patients with Parkinson's disease is decreased compared to membranes prepared from age- and sex-matched controls. These data suggest that [3H]GBR-12935 binds in a sodium-dependent fashion to the dopamine transport complex in human brain and that specific binding is decreased by a pathological degeneration of dopaminergic neurons to the caudate nucleus.     

Preconditioning with a Novel Metallopharmaceutical NO Donor in Anesthetized Rats Subjected to Brain Ischemia/Reperfusion

Rut-bpy is a novel nitrosyl–ruthenium complex releasing NO into the vascular system. We evaluated the effect of Rut-bpy (100 mg/kg) on a rat model of brain stroke. Forty rats were assigned to four groups (Saline solution [SS], Rut-bpy, SS+ischemia–reperfusion [SS+I/R] and Rut-bpy+ischemia–reperfusion [Rut-bpy+I/R]) with their mean arterial pressure (MAP) continuously monitored. The groups were submitted (SS+I/R and Rut-bpy+I/R) or not (SS and Rut-bpy) to incomplete global brain ischemia by occlusion of the common bilateral carotid arteries during 30 min followed by reperfusion for further 60 min. Thirty minutes before ischemia, rats were treated pairwise by intraperitoneal injection of saline solution or Rut-bpy. At the end of experiments, brain was removed for triphenyltetrazolium chloride staining in order to quantify the total ischemic area. In a subset of rats, hippocampus was obtained for histopathology scoring, nitrate and nitrite measurements, immunostaining and western blotting of the nuclear factor- κB (NF-κB). Rut-bpy pre-treatment decreased MAP variations during the transition from brain ischemia to reperfusion and decreased the fractional injury area. Rut-bpy pre-treatment reduced NF-κB hippocampal immunostaining and protein expression with improved histopathology scoring as compared to the untreated operated control. In conclusion, Rut-bpy improved the total brain infarction area and hippocampal neuronal viability in part by inhibiting NF-κB signaling and helped to stabilize the blood pressure during the transition from ischemia to reperfusion.     

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.     

Determination of p-trifluoromethylphenol, a metabolite of fluoxetine, in tissues and body fluids using an electron-capture gas chromatographic procedure

An electron-capture gas chromatographic procedure was developed for the analysis of p-trifluoromethylphenol, an O-dealkylated metabolite of fluoxetine, in biological samples. A basic extraction of the biological sample was employed, followed by derivatization with pentafluorobenzenesulfonyl chloride. The internal standard, 2,4-dichlorophenol, was added to all samples used in the procedure to aid in quantitation. The practical limit of detection (signal-to-noise ratio>3) for p-trifluoromethylphenol was <5 ng/ml in human plasma samples, <10 ng/g of rat brain tissue, <25 ng/g of rat liver tissue and <25 ng/ml in human and rat urine samples. In the rat, the levels of free p-trifluoromethylphenol in the liver were 10-fold higher than those in the brain, and a substantial amount was excreted in the urine. Human urine samples contained levels of free p-trifluoromethylphenol approximately 30-fold higher than those found in human plasma samples. The procedure described is useful for the detection and quantitation of free p-trifluoromethylphenol in humans and rats treated with fluoxetine.