HSP70 Immunoreactivity Research Articles

Transpupillary thermotherapy (TTT) with short duration laser exposures induce heat shock protein (HSP) hyperexpression on choroidoretinal layers.

To assess a choroidal heat shock protein hyperexpression after transpupillary thermotherapy (TTT) performed with exposures shorter than 60 seconds. Nine male pigmented rabbits were anesthetized and TTT was performed on their right eye with a 810 nm diode laser (Iridis, Quantel-Medical (France)) (spot size: 1.3 mm). Three exposure durations (60, 30, or 15 seconds) were used with three ranges of power for each duration ("high," "mild," or "low"). A series of laser impacts was delivered to the posterior pole of the retina. Left eyes were used as controls. Twenty-four hours after laser irradiation, the animals were killed and histological study was performed on chorioretinal layers. Tissue samples were fixed in formalin and embedded in paraffin. A monoclonal antibody was used to detect Hsp70 immunoreactivity (mouse IgGl, SPA-810, Stress Gen, Victoria, BC, Canada), followed by a biotinylated goat anti-mouse antibody (Dako, Glostrup, Denmark), revealed by the avidin-biotin complex (Vectastain kit, Vector Laboratries, Burlingame, CA, USA) and the AEC chromogen. Retinal structures were further identified by HES coloration. During the experiments, the laser spots were not visible except for the strongest "high" powers for each exposure duration, where a whitening was discernable at the end of the laser exposures. A strong HSP70 immunoreactivity was detected in choroidal, non-pigmented cells for laser exposures lasting 60, 30, or 15 seconds with "mild" laser powers. On the contrary, rare HSP hyperexpression was detected with "high" or "low" laser powers lasting 60, 30, or 15 seconds. No HSP-70 immunoreactivity was detected on control eyes nor outside of the irradiated zones of treated eyes. Transpupillary laser irradiation lasting 15, 30, or 60 seconds induces an hyperexpression of HSP on choroidal layers. This could be a basis for the use of TTT with "short" laser exposures.

Hyperthermic pre-conditioning protects retinal neurons from N-methyl- d-aspartate (NMDA)-induced apoptosis in rat

Glutamate-induced excitotoxicity is associated with a selective loss of retinal neurons after retinal ischemia and possibly in glaucoma. Since heat shock protein (HSP) 70 is known to play a protective role against ischemic neuronal injury, which is also linked to excitotoxicity, we studied the expression of inducible (HSP72) and constitutive (HSC70) forms of HSP70 in apoptosis of retinal ganglion cells (RGCs) after intravitreal injection of 8 nmoles N-methyl- d-aspartate (NMDA), a glutamate receptor agonist. Approximately 18 h after NMDA injection, there were increased numbers of TUNEL-positive cells and cells with elevated HSP72 immunoreactivity in the retinal ganglion cell layer (RGCL), but there were no noticeable changes in HSC70 immunoreactivity. These HSPs positive cells were also Thy-1 positive, a marker for RGCs. Hyperthermic pre-conditioning, which is known to induce HSPs, given 6 or 12 h prior to NMDA injection ameliorated neuronal loss in the RGCL as counted 7 days after NMDA injection but pre-conditioning at 18 h prior to NMDA injection did not have any ameliorative effect. Quercetin, an inhibitor of HSP synthesis, abolished the ameliorative effect of hyperthermic pre-conditioning. Pre-conditioning elevated HSP72 but not HSC70 immunoreactivity and reduced the number of TUNEL-positive cells in the RGCL at 18 h. Our results suggest that intravitreal injection of NMDA induces an up-regulation of HSP72 in a time-dependent manner but not HSC70 in RGCs, indicating a stress response of HSP72 in RGCs and other inner retinal neurons after exposure to NMDA. Hyperthermic pre-conditioning given within a therapeutic window is neuroprotective to the retina against NMDA-induced excitotoxicity, likely by inhibiting apoptosis through the modulation of HSP72 expression.

HSP70 constitutive expression in rat central nervous system from postnatal development to maturity.

We studied the level of the basal (constitutive) HSP70 expression (inducible and constitutive forms) in the central nervous system (CNS) of male and female rats from the postnatal period to maturity. HSP70 levels were analyzed by immunoblotting in five different areas (cortex, hippocampus, hypothalamus, cerebellum, and spinal cord). The highest levels of HSP70 were found in juvenile rats and decreased progressively until reaching baseline levels between 2 and 4 months. A slight and nonsignificant increase in aged (2-year-old) rats compared with adult subjects was observed in some cerebral areas (cerebral cortex, hippocampus, and cerebellum). In the first weeks of postnatal development, HSP70 immunoreactivity was distributed throughout CNS sections and no specific immunopositive cells could be clearly determined. In adult animals, strong immunostaining was observed in some large neurons (Purkinje neurons and mesencephalic and spinal cord motor neurons), some perivascular and subpial astrocytes, and ependymocytes. Immunoelectron microscopy revealed that HSP70 in these cells is located in the perinuclear area and in mitochondria, rough endoplasmic reticulum, and microtubules. In neurons, strong immunolabeling was also observed in synaptic membranes. The postnatal time course of HSP70 levels and the location and size of HSP70-immunopositive cells suggest that HSP70 constitutively expressed in the rat CNS may be mainly determined by the degree of development and metabolic activity of the neural cells.

The Effect of Neonatal Nerve Injury on the Expression of Heat Shock Proteins in Developing Rat Motoneurones

The expression of the heat shock proteins hsp27 and hsp70 was examined in the spinal cord and sciatic nerves of developing rats. Using immunohistochemistry, we found that hsp27 is present in many motoneurones at birth. With development, the intensity of staining increases, reaching adult levels by 21 days, when all sciatic motoneurones express hsp27. In the sciatic nerve, hsp27 is strongly expressed throughout postnatal development. In contrast, hsp70 immunoreactivity in motoneurones and the sciatic nerve is weak at birth and does not change with development. The expression of heat shock proteins has been shown to increase in cells under conditions of stress, where they have beneficial effects on cell survival. The effect of neonatal nerve injury on hsp27 and hsp70 expression was also examined in this study. Four days after injury, staining for hsp27 increases in motoneurones, whereas hsp70 does not change. However, there is a significant increase in hsp70 staining in glial cells surrounding the injured motor pool, predominantly in astrocytes. Since neonatal nerve injury induces apoptotic motoneurone death, we also studied the co-expression of hsp27 with markers of apoptosis. No hsp27-positive motoneurones were found to be apoptotic, as assessed by both TUNEL and caspase-3 immunoreactivity. Therefore, it is possible that the upregulation of hsp27 observed in injured motoneurones may play a role in protecting motoneurones from apoptotic cell death following nerve injury.

Targeted hsp70.1 disruption increases infarction volume after focal cerebral ischemia in mice.

Heat-shock proteins (HSPs) are highly conserved proteins that are induced by a variety of stresses. HSP70 is a 70-kDa HSP family known to have cytoprotective effects against various insults. The role of HSP70 in cerebral ischemia remains to be elucidated in vivo. To investigate the effect of reduced HSP70 levels on cerebral ischemia, focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery was induced in hsp70.1 knockout mice. The expressions of hsp70.1 and hsp70.3 mRNAs and HSP70 protein were determined, and infarction volumes were measured and compared. Northern blots confirmed the absence of hsp70.1 mRNA expression in the knockout mice. The mean infarction volume was significantly larger in hsp70.1 knockout mice (92.5+/-8.3 mm(3)) than in the wild-type mice (59.3+/-8.9 mm,(3) P<0.001). Western blots showed increased HSP70 expression in the ischemic hemisphere in both knockout and wild-type mice, but HSP70 expression levels in knockout mice were significantly lower than those in their wild-type littermates. Immunohistochemistry did not show any significant differences between the knockout and wild-type animals and showed increased HSP70 immunoreactivity in the ischemic hemisphere, with predominance in the cerebral cortex, especially in the penumbra. Our results suggest that hsp70.1 plays an important role in the early protection of the brain, at least after acute focal cerebral ischemia in mice.

Transient occurrence of 27 kDa heat-shock protein in the terminal tubule cells during postnatal development of the rat submandibular gland.

It has been suggested that the 27 kDa heat-shock protein (Hsp27) plays a role at crucial cellular checkpoints for proliferation, apoptosis, and differentiation. We examined the immunolocalization of Hsp27 in the rat submandibular gland during postnatal development, wherein acinar cells proliferate and differentiate at earlier postnatal periods. At 2 weeks of age, weak Hsp27 immunoreactivity was distributed diffusely over all gland components. At 3 weeks, Hsp27 immunoreactivity disappeared in most parts of the acini and ducts, but was intensely accumulated in a small cell population located in the acinar center. This population was composed mostly of terminal tubule (TT) type I cells. At 4 weeks, the Hsp27-immunopositive cell population in the acinar center was composed primarily of immature (type II) acinar cells, partly of immature (granulated) intercalated duct (ID) cells, and occasionally of apoptotic cells. After 5 weeks, all acinar components became mature and were no longer immunoreactive for Hsp27. When acinar cell differentiation was accelerated by administration of isoproterenol to 3-week-old rats for 7 days, the number of Hsp27-positive cells was significantly lower than in the control gland at 4 weeks, confirming that Hsp27 expression is downregulated in mature acinar cells. These results suggest that at around 3-4 weeks in postnatal development, the centroacinar TT cells stop proliferating and begin to differentiate into acinar and ID cells, and occasionally undergo apoptosis. Hsp27 is transiently expressed in the centroacinar TT cells during this critical period, and thus may play a role in their differentiation into the immediate descendants.

Heat shock protein expression in the transplanted human kidney.

Heat shock proteins (HSPs) have been shown to represent potential target molecules for T-cell-mediated allograft rejection in heart and kidney transplants. In the present study, we therefore investigated the expression of HSP subtypes 60, 72, and 73 in normal kidneys and qualitative and/or quantitative changes in rejected renal allografts. Six normal kidney tissue specimens, three biopsies from patients with minimal change nephritis, as well as 37 biopsies and eight transplant nephrectomy specimens of patients with renal allograft rejection were studied. Type and severity of rejection were assessed according to the Banff classification. Immunohistochemical demonstration of HSP expression was performed using specific monoclonal antibodies after wet autoclave antigen retrieval on sections from either Carnoy-fixed (biopsies) or formalin-fixed (transplant nephrectomies) and paraffin-embedded tissue. The expression was scored in a semiquantitative manner. All three subtypes were found to be constitutively expressed in normal kidney tissue and in noninflammatory minimal change nephritis, albeit with a characteristic compartmental and cellular distribution. Rejection resulted in a higher immunohistochemical scoring for all three HSP subtypes in compartments in which they were normally present; in addition, a de novo expression of HSP60 was found in the vascular compartment and, moreover, infiltrating mononuclear cells were strongly immunoreactive for HSP60 and HSP73. Only quantitative differences were observed for HSP72 immunoreactivity. These results indicate that rejection episodes are paralleled by an increased but differential expression of HSPs in the glomerular, tubular, and vascular compartments of the kidney. This enhancement as well as the de novo appearance of HSP60 on vascular endothelial cells might explain the presence of HSP-reactive T lymphocytes in rejected allografts.

Alanyl-Glutamine Dipeptide Does Not Affect Hemodynamics despite a Greater Increase in Myocardial Heat Shock Protein 72 Immunoreactivity in Endotoxemic Sheep

The possible beneficial effect of supplemental glutamine (Gln) in critically ill patients has been suggested to be mediated by the induction of the cytoprotective heat shock proteins (HSP)32 and HSP72. There is evidence that HSP72 and HSP32 have opposite effects on the hemodynamic situation during endotoxemia. Therefore, the effect of Gln supplementation on the cardiovascular system is not clear. We investigated the effect of alanyl-Gln (Ala-Gln) dipeptide on cardiovascular function in healthy and endotoxemic sheep. Ten sheep catheterized for chronic studies received Ala-Gln 700 mg/(kg x d) [equal to 470 mg/(kg x d)Gln] on 4 consecutive days, and 10 sheep received NaCl (9 g/L) as the control solution. On d 4, four sheep of each group were killed and myocardial samples were taken for immunohistochemistry. The remaining sheep received a continuous infusion of endotoxin [Salmonella typhosa, 10 ng/(kg x min)]. Hemodynamic parameters were measured before application of Ala-Gln or the control solution, and during endotoxemia. Myocardial HSP72 immunoreactivity was determined by immunohistochemistry. After 24 h of endotoxemia, the sheep exhibited a hyperdynamic circulation. No difference was found in the hemodynamic parameters between treatment and control group. Ala-Gln treated sheep had a greater increase in myocardial HSP72 immunoreactivity compared with controls after (P < 0.05) but not before endotoxemia. In summary, Ala-Gln increased HSP72 immunoreactivity after endotoxemia, but did not alter hemodynamic parameters. Thus, Ala-Gln supplementation does not seem to aggravate the hyperdynamic circulation in endotoxemic shock.

Infarct tolerance accompanied enhanced BDNF-like immunoreactivity in neuronal nuclei

A prolonged period (48 h) of cortical spreading depression (CSD) induced resistance against severe focal cerebral ischemia (infarct tolerance), however, the mechanism behind this is unknown. The infarct tolerance was a transient phenomenon; the resistance increased linearly for the initial 12 days, peaking from 12 to 15 days after a preconditioning of CSD, and was decreased thereafter. This study examined the time course of brain-derived neurotrophic factor (BDNF), heat shock protein (hsp)27 and 70, and glial fibrillary acidic protein (GFAP) expressions after CSD in the brain. Immunohistochemical expression of BDNF, hsp27, hsp70, or GFAP following a prolonged period of CSD induced by KCl-infusion, or following NaCl-infusion was analyzed by regional densitometry for 24 days in the rat neocortex. In addition, BDNF protein was measured quantitatively by two-site ELISA assay in the neocortex ( n=6 at each time point). The GFAP expression was elevated in astrocytes (compared to the normal level of immunodensity) during the period peaking on day 3–6 following the CSD. The hsp27 immunoreactivity was also elevated in astrocytes from day 1 to 12 peaking on day 1 and 6, but there was no expression of hsp70 during the period following CSD. The immunoreactivity for BDNF was elevated in neurons from day 0 to 18 peaking on day 1 and 6. The protein levels of BDNF in the neocortex were significantly elevated from day 0 to 12 peaking on days 0 and 6 (compared to the normal level) ( P<0.05). Using a laser-scanning confocal imaging system, the BDNF-like immunoreactivity in neuronal nuclei was found to increase linearly peaking on day 12, which correlated well with the development of infarct tolerance. The intranuclear increase in BDNF-like protein might contribute to the induction of infarct tolerance in the brain.

Induction by Synaptic Zinc of Heat Shock Protein-70 in Hippocampus after Kainate Seizures

Following seizures, heat shock protein (HSP)-70 is induced in various brain regions. Since zinc that can induce HSP-70 in various cell systems is enriched in certain glutamatergic terminals and translocates to postsynaptic neurons with seizures, we examined the possibility that HSP-70 induction in the epileptic brain is mediated by synaptic zinc. Adult rats were injected intraperitoneally with kainate to induce seizures. Seizures were halted 3 h after the kainate administration by the injection of phenytoin. Staining of brain sections with zinc-specific fluorescent dye TFL at 24 h after the kainate injection revealed a one-to-one correlation between dense TFL fluorescence and acidophilic neuronal degeneration in the hippocampus. Subsequent staining with anti-HSP-70 antibody, however, revealed that more numerous neurons than degenerating neurons exhibited HSP-70 immunoreactivity. Most of the HSP-70(+) neurons were not stained with acid fuchsin but exhibited mild zinc fluorescence in the cytoplasm. Intraventricular injection of CaEDTA attenuated neuronal death as well as the HSP-70 induction in a dose-dependent manner. Supporting the specificity of zinc rather than calcium as the inducer of HSP-70 in neurons, exposure to zinc but not to a calcium ionophore or excitotoxins increased expression of HSP-70 mRNA and protein in cultured cortical neurons. The present results suggest that not only selective neuronal death, but also HSP-70 induction in neurons after seizures, is mediated by the translocation of endogenous synaptic zinc.

Metallothionein and heat shock protein expression during acute liver injury and regeneration in rats.

Metallothioneins (MT) are cytosolic proteins rich in cysteine which play a physiological role in metal ion homeostasis. Heat shock proteins (HSPs) are expressed in various organs in response to different stress stimuli. The purpose of the present study was to examine the intrahepatic distribution of MT and HSP-27, -70 and -90 in two different experimental models of acute liver injury and regeneration, induced by either thioacetamide, or carbon tetrachloride administration in male Wistar rats. Toxicological endpoints and markers of hepatocellular regeneration were assessed at various time points following toxin administration. The enzymatic activities of aspartate and alanine aminotransferases in serum, and histological findings in the liver were used to estimate toxin-induced injury. Tritiated thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index were used to estimate liver regeneration. MT and HSPs were detected immunohistochemically. At the time of maximum liver injury, moderate MT and intense HSPs expression was prominent in hepatocytes in the vicinity of necrotic areas. At the time of maximum hepatocellular proliferation, intense MT and HSP-90 staining was evident in all hepatocytes, while at the same time, mild HSP-27 and HSP-70 immunoreactivity was noted. Our findings indicate that the differential distribution of MT and HSPs in the liver after toxin-induced injury, in common with the observed pattern of staining, reflect liver proliferating capacity.

Estrogen receptor gene expression and its relation to the estrogen-inducible HSP27 heat shock protein in hormone refractory prostate cancer.

The recent discovery of the classical estrogen receptor alpha (ERalpha) in androgen-insensitive prostate cancer has shed new light on the role of estrogens in endocrine therapy failure. To get more information on downstream events of estrogen signaling in these tumors, we investigated the relation between ERalpha gene expression, and the estrogen-inducible heat shock protein HSP27 in recurrent prostatic adenocarcinomas. Palliative transurethral resection specimens from 50 patients with androgen-insensitive disease were submitted for study. Messenger RNA in situ hybridization for the ERalpha and immunohistochemistry of the HSP27 protein were performed on adjacent sections of an equal number of prostate cancer tissue with and without ERalpha protein expression. Cancerous lesions lacking the nuclear ERalpha at the protein level revealed ERalpha mRNA expression in 15 of 25 cases (60%). A coordinate expression of ERalpha mRNA and HSP27 was observed in 33 of 40 cases (83%), although a significant correlation between ERalpha protein and HSP27 expression was not obtained. Conversely, 90% of neoplastic lesions without detectable levels of ERalpha mRNA and protein also lacked HSP27 immunoreactivity. ERalpha gene expression at the mRNA level significantly correlated with the immunoprofile of the estrogen-inducible HSP27 protein in androgen-insensitive prostatic adenocarcinomas. This may indicate that these tumors harbor functional active estrogen receptors promoting transcriptional activity of the HSP27 gene. Determination of the receptor status by immunohistochemistry is unable to identify neoplastic lesions with established ERalpha mRNA expression in a substantial number of cases. HSP27 may be an additional surrogate biomarker for estrogen-regulated growth in androgen-insensitive prostate cancer.

Hyperthermic induction of the 27-kDa heat shock protein (Hsp27) in neuroglia and neurons of the rat central nervous system.

The 27-kDa heat shock protein (Hsp27) is constitutively expressed in many neurons of the brainstem and spinal cord, is strongly induced in glial cells in response to ischemia, seizures, or spreading depression, and is selectively induced in neurons after axotomy. Here, the expression of Hsp27 was examined in brains of adult rats from 1.5 hours to 6 days after brief hyperthermic stress (core body temperature of 42 degrees C for 15 minutes). Twenty-four hours following hyperthermia, Western blot analysis showed that Hsp27 was elevated in the cerebral cortex, hippocampus, cerebellum, and brainstem. Immunohistochemistry for Hsp27 revealed a time-dependent, but transient, increase in the level of Hsp27 immunoreactivity (Hsp27 IR) in neuroglia and neurons. Hsp27 IR was detected in astrocytes throughout the brain and in Bergmann glia of the cerebellum from 3 hours to 6 days following heat shock. Peak levels were apparent at 24 hours, gradually declining thereafter. In addition, increases in Hsp27 IR were detected in the ependyma and choroid plexus. Hyperthermia induced Hsp27 IR in neurons of the subfornical organ and the area postrema within 3 hours and reached a maximum by 24 hours with a return to control levels 4-6 days after hyperthermia. Specific populations of hypothalamic neurons also showed Hsp27 IR after hyperthermia. These results demonstrate that hyperthermia induces transient expression of Hsp27 in several types of neuroglia and specific populations of neurons. The pattern of induced Hsp27 IR suggests that some of the activated cells are involved in physiological responses related to body fluid homeostasis and temperature regulation.

Immunohistochemical localization of heat shock protein 70 in the human medulla oblongata in forensic autopsies

Heat shock protein 70 (hsp70) can be induced under various stresses in experimental animals. We investigated hsp70 immunoreactivity in the human medulla oblongata in forensic autopsies. Hsp70 immunoreactivity was observed in the cytoplasm of some neurons in the hypoglossal nucleus (XII), the dorsal motor nucleus of the vagal nerve (X), the lateral cuneate nucleus (Cun), and the inferior olive (Oli). Neurons with positive hsp70 immunoreactivity were statistically significantly fewer in the Oli than in the XII, X, and Cun. There was no statistically significant correlation between the AMI (the antemortem interval between the onset of injury and death) or PMI (the postmortem interval between death and autopsy), and the percentage of positive cytoplasmic hsp70 immunoreactivity in any of the nuclei studied. Age had a statistically significant negative correlation with the percentage of positive hsp70 immunoreactivity in the Oli. The percentages of positive hsp70 immunoreactivity in the XII and Cun were statistically significantly lower in burn cases than in other cases. Therefore, the induction of hsp70 immunoreactivity in the medulla oblongata may not reflect the duration of stress in the AMI, but may reflect the regional (nuclei) and conditional (burns) differences in autopsy specimens.

Hsp72 antigen expression in the proliferative compartment of involved psoriatic epidermis.

Oxidative damage to growth regulatory proteins has been implicated in the aetiology of psoriasis. However, the transient synthesis of heat shock proteins has been shown to protect cells against the adverse effects of oxidative and other forms of physiological stress. This study has used an hsp72 monoclonal antibody to measure inducible 72 kDa heat shock protein expression in heat stressed normal human skin and established plaque psoriasis. Hsp72 was detected in the basal and suprabasal layer cells of heat-stressed normal skin, and in 12 involved psoriasis lesions. Hsp72 expression was not detected in unstressed normal skin or in 12 cases of uninvolved psoriasis. Immunoprecipitation and Western blotting of cell lysates from heat stressed normal skin and involved psoriasis lesions confirmed the presence of a 72 kDa polypeptide with hsp72 immunoreactivity. The MIB-1 monoclonal antibody was used to determine the proliferative fraction of normal and involved psoriastic epidermis. The Ki67 antigen was localised to the nuclei of basal and suprabasal layer cells of normal and involved psoriatic epidermis. Involved psoriatic epidermis contained a higher number of proliferating keratinocytes when compared with normal skin. The study has also demonstrated a strong correlation between hsp72 expression and keratinocyte proliferation in involved psoriatic epidermis (r=0.864, p<0.001). We believe that the 72 kDa inducible heat shock protein performs a protective function in the proliferative compartment of normal and involved psoriatic skin.

Induction of heat shock protein 70 in the rat brain following intracisternal infusion of autologous blood: evaluation of acute neuronal damage.

Investigation into a potential treatment for the acute period following onset of spontaneous subarachnoid hemorrhage (SAH) is hampered by the lack of a standardized experimental model. For that purpose the authors elaborated on a small-animal model in which computer-controlled intracisternal blood infusion is used and investigated whether this model can reliably reproduce acute neuronal injury after SAH. Whole autologous blood (blood-infused group) or isotonic saline (control group) was infused into the cisterna magna or olfactory cistern of rats. The infusions decreased exponentially during a 5-minute period. Throughout the infusion period, intracranial pressure (ICP) was monitored. Neuronal injury was quantified by observing tissue immunoreactivity to a 70-kD heat shock protein (HSP70) and comparing this with the tissue's reaction to hematoxylin and eosin staining. On Days 1, 3, and 5, the CA1, CA3, and dentate gyrus regions of the hippocampus were analyzed, respectively. During saline infusion ICP increased within seconds beyond 80 mm Hg and afterward decreased in accordance with the infusion rate. During the infusion of blood, the same initial pressure peak was found, but the ICP remained increased beyond this pressure level throughout the 5-minute infusion period. The HSP70 immunoreactivity in the saline-infused group was found only on Day 1 in the CA1 region and the dentate gyrus, but not in the CA3. After injection of whole blood, there was HSP70-positive staining in the CA1, CA3, and dentate gyrus regions throughout the observation period. The controlled cisternal infusion of blood caused neuronal injury that resembled that of previous experimental models that produce SAH by rupture of intracranial vessels with endovascular techniques. Unlike those experiments, the intracisternal infusion technique presented by the authors provides more standardized bleeding with regard to ICP, the volume of subarachnoid blood, and the extent of acute cellular injury.

Response of carp central nervous system to hyperammonemic conditions: an immunocytochemical study of glutamine synthetase (GS), glial fibrillary acidic protein (GFAP) and 70 kDa heat-shock protein (HSP70)

Glutamine synthetase (GS), glial fibrillary acidic protein (GFAP) and 70 kDa heat-shock protein (HSP70) expression were studied in neural tissue of adult carp subjected to hyperammonemic conditions using immunocytochemical methods (immunoblotting, dot blotting, ELISA and immunocytochemistry). Carps were subjected to 1, 2 and 4 mM ammonium acetate for 7, 15, 30 and 60 days since the mortality rate was 100% at 8 mM within 2 days. The mortality rate was 27% at 4 mM. A significant dose-dependent induction of GS was observed in hyperammonemic brains. GS immunoreactivity was located in radial astrocytes and some ependymal cells. Significant increments of GFAP were observed at 30 days for 1 mM, at 15 and 30 days for 2 mM, and at 30 and 60 days for 4 mM; a heterogeneous response to hyperammonemia was observed in different brain regions. Significant increments in HSP70 were observed in hyperammonemic brain (30 days, 4 mM). The neuronal pattern of HSP70 immunoreactivity under control conditions changed to an ependymal pattern under hyperammonemic conditions. These results suggest that the main effect of hyperammonemia was GS induction in neural tissue. GFAP induction could be an effect of astroglial activation and HSP70 induction could be related with de novo protein synthesis.

Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70-kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150-160). The 2A4(+)ORNs are dispersed through zones II-IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress-inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters.

C-Fos, Jun D and HSP72 immunoreactivity, and neuronal injury following lithium-pilocarpine induced status epilepticus in immature and adult rats

In order to follow the maturation-related evolution of neuronal damage, cellular activation and stress response subsequent to Li-Pilo seizures in the 10- (P10), 21-day-old (P21) and adult rat, we analyzed the expression of the c-Fos protein as a marker of cellular activation, HSP72 immunoreactivity as the stress response and silver staining for the assessment of neuronal damage in 20 selected brain regions. The early wave of c-Fos measured at 2 h after the onset of seizures was present in most structures of the animals at the three ages studied and particularly strong in the cerebral cortex, hippocampus and amygdala. The late wave of c-Fos measured at 24 h after the onset of seizures and that was shown to correlate to neuronal damage was absent from the P10 rat brain, and present mainly in the cerebral cortex and hippocampus of P21 and adult rats. The expression of the stress response, assessed by the immunoreactivity of HSP72 at 24 h after the seizures was absent from the P10 rat brain and present in the entorhinal cortex, amygdala, hippocampus and thalamus of P21 and adult rats. The expression of Jun D at 24 h after the seizures was discrete and present in most brain regions at all ages. Neuronal injury assessed by silver staining at 6 h after the onset of seizures was very discrete in the brain of the P10 rat and limited to a few neurons in the piriform and entorhinal cortices. In older animals, marked neuronal degeneration occurred in the cerebral cortex, amygdala, hippocampus, lateral septum and thalamus. Thus the immediate cell activation induced by lithium-pilocarpine seizures which is present at all ages translates only into a late wave of c-Fos and the expression of HSP72 in P21 and adult animals in which there will be extensive cell damage.

Expression of 70-kDa heat shock protein in oral lesions: marker of biological stress or pathogenicity.

We showed differential expression of HSP70 during oral tumorigenesis. The precise functional role of HSP70 overexpression in the pathogenesis of betel and tobacco related oral cancer remains to be determined. To evaluate the utility of HSP70 as an indicator of the biological stress experienced by tumour cells or the malignant potential of oral epithelial lesions and predicting clinical outcome, its expression was assessed in different stages of oral carcinogenesis by immunohistochemical analysis and correlated with clinicopathological parameters. Overexpression of HSP70 protein was observed in 38 of 64 (59%) dysplastic lesions and 92 of 125 (74%) oral squamous cell carcinomas (SCCs) which included 76 of 105 cases (72%) of primary oral SCCs and 16 of 20 (80%) of recurrent oral SCCs. A significant correlation of HSP70 expression was observed with severity of dysplasia (P = 0.0006767), poor histological differentiation of primary tumours (P = 0.0184348), increase primary tumour size (P = 0.0221103) and consumption of betel and tobacco (P < 0.01). Follow-up studies showed that in patients with premalignant lesions the median transition time (premalignancy to malignancy) was significantly shorter in HSP70 overexpressing cases than those showing basal level of HSP70 (P = 0.012). Oral cancer patients with elevated levels of HSP70 showed decreased median disease-free survival time (no recurrence/metastasis) than those showing basal HSP70 immunoreactivity (P = 0.0246). The results suggest that HSP70 expression may not be a mere marker of biological stress but may also be implicated in the pathogenesis of oral cancer.