Immuno-electron-microscopic study of the prolactin cells in the pituitary gland of male Wistar rats during aging

Summary Prolactin cells were identified by means of immunocytochemistry with protein-A gold as a marker on ultrathin sections of the pituitary gland of young (3–4 months), middle-aged (16–19 months), and aged (26–30 months) male Wistar rats. Point-counting volumetry revealed that the prolactin (PRL) cell-volume density in middle-aged rats was significantly increased in comparison to the volume densities in young and aged rats. Within the PRL-cell population, four types of PRL cells were distinguished on the basis of the shape and size of their secretory granules. During aging, dramatic changes occurred in the relative volumes of the four cell types. The volume percentage of cells with round granules (type I, granule diameter 150–250 nm, and type IIA, granule diameter 250–350 nm) increased from ±30% in young rats to ±90% in old rats. The volume percentage of cells with round and polymorphic granules (type IIB; granule diameter 350–400 nm and type III; granule diameter 500–600 nm) decreased from ±70% in young rats to ±7% in old rats. Age-related changes in serum PRL levels were not found. It is concluded that although during the life span of the male Wistar rat considerable changes in PRL-cell volume densities and in the ratios of PRL-cell types occur serum, PRL levels remain more or less constant.     

Glutamate-Glutamine Cycle and Aging in Striatum of the Awake Rat: Effects of a Glutamate Transporter Blocker

This study investigated the effects of aging on the actions of a specific glutamate reuptake blocker, L-trans-pyrrolidine-2, 4-dicarboxylic acid (PDC), in extracellular glutamate and glutamine in striatum of the awake rat. Microdialysis experiments were performed on young (2–3 months), middle-aged (12–14 months), aged (27–32 months) and very aged (37 months) male Wistar rats. Local infusion of PDC (1–4 mM) in striatum increased the dialysate concentration of glutamate and decreased dialysate concentration of glutamine in all the age-groups. In young rats, decreases of dialysate glutamine were correlated with increases of dialysate glutamate. The same profile glutamine/glutamate as in young rats was found in middle-aged, aged and very aged rats, which suggests that the action of glutamate on the glutamate-glutamine cycle in striatum of the awake rat is not modified as a consequence of aging. We also found a significant correlation between the increases of glutamate produced by PDC and the basal dialysate concentration of glutamine, a relationship that did show a significant change with age. Although the significance of this latter finding remains to be elucidated, it may be important to understand the changes in glutamate-glutamine cycle during aging.     

The effect of age on protein composition of rat cerebral microvessels

The effect of age on protein composition of cerebral microvessels was investigated by examining the content of glycosylation endproducts in cerebral microvessels isolated from young (3–6 month old), intermediate age (18 month) and aged (24–26 month old) Fischer 344 male rats and by quantitating various protein spots identified with two dimensional (2D) electrophoresis. The results indicate that aging in rats is not associated with significant increase in glycosylation of microvessel proteins. Of the 26 proteins in cerebral microvessels identified on the 2-D gel, ten showed significant age-related changes (p<0.0004) and in two of these the changes were significant as early as 18-months of age. A large acidic protein with a molecular weight of 144,000 and isoelectric point (pI) of 5.4 (Spot #1) was found only in aged rats. The results indicate that aging is associated with significant quantitative changes in protein composition of cerebral microvessels. It is possible that Spot #1 may be a novel biochemical marker of aging blood-brain barrier.     

Effects of estrogens on choline-acetyltransferase immunoreactivity and GAP-43 mRNA in the forebrain of young and aging male rats

1. Previous work demonstrated that estradiol (E₂) treatment prevented the abnormal response to stress and the reduction of glucocorticoid receptors (GR) in hippocampus from aging male rats. The mechanisms originating these effects were unknown.2. In the present work, we investigated the E₂ effects on the cholinergic, growth-associated protein (GAP-43) expressing neurons of the medial septum (MS) and vertical limb of diagonal band of Broca (VDB). These areas project to the hippocampus, and may be involved in the mentioned E₂ effects in aging animals. Therefore, the response to E₂ of choline-acetyltransferase (ChAT) in neurons and cell processes and GAP-43 mRNA as a marker of neurite outgrowth was studied in young and old male rats.3. Young (3–4 months) and old (18–20 months) male Sprague-Dawley rats remained untreated or were implanted s.c. with a 14 mg pellet of E₂ benzoate during 6 weeks. We used immoucytochemistry to determine ChAT and isotopic in situ hybridization to analyze GAP-43 mRNA expression.4. Aging males showed a reduction in the number and length of ChAT-immunoreactive cell processes, but not in the number of positive neurons in MS and VDB. E₂ reverted both parameters in old rats to levels of young animals. Regarding basal levels of GAP-43 mRNA, they were similar in old and young animals, but E₂ treatment up-regulated GAP-43 mRNA expression in MS and VDB of old animals only.5. Our data suggest that prolonged E₂ treatment may affect hippocampal function of aging male rats by regulating in part the plasticity of cholinergic, GAP-43 expressing neurones of the basal forebrain. Without discarding a direct E₂ effect on the limbic tissue, effects on the cholinergic system may have a pronounced impact on the neuroendocrine and stress responses of the aging hippocampus.     

Anti-Peroxynitrite Treatment Ameliorated Vasorelaxation of Resistance Arteries in Aging Rats: Involvement with NO-sGC-cGKs Pathway

Declined vasorelaxation function in aging resistance arteries is responsible for aging-related multiple organ dysfunctions. The aim of the present study is to explore the role of peroxynitrite (ONOO^-) in aging resistance arterial vasorelaxation dysfunction and the possible mechanism. In the present study, young (3–4 months olds) and aging (20 months olds) male SD rats were randomized to receive vehicle (Saline) or FeTMPyP (ONOO^- scavenger) for 2 weeks. The vasorelaxation of resistance arteries was determined in vitro; NOx level was tested by a colorimetric assay; the expression of nitrotyrosine (NT), soluble Guanylate Cyclase (sGC), vasodilator-stimulated phosphoprotein (VASP), phosphorylated VASP (P-VASP) and cGMP in resistance arteries were detected by immunohistochemical staining. In the present study, endothelium-dependent dilation in aging resistance arteries was lower than in those from young rats (young vs. aging: 68.0%±4.5% vs. 50.4%±2.9%, P<0.01). And the endothelium-independent dilation remained constant. Compared with young rats, aging increased nitrative stress in resistance arteries, evidenced by elevated NOx production in serum (5.3±1.0 nmol/ml vs. 3.3±1.4 nmol/ml, P<0.05) and increased NT expression (P<0.05). ONOO^- was responsible for the vasorelaxation dysfunction, evidenced by normalized vasorelaxation after inhibit ONOO^- or its sources (P<0.05) and suppressed NT expression after FeTMPyP treatment (P<0.05). The expression of sGC was not significantly different between young and aging resistance arteries, but the cGMP level and P-VASP/VASP ratio (biochemical marker of NO-sGC-cGKs signaling) decreased, which was reversed by FeTMPyP treatment in vivo (P<0.05). The present study suggested that ONOO^- mediated the decline of endothelium-dependent vasorelaxation of aging resistance arteries by induction of the NO-sGC-cGKs pathway dysfunction.     

Cytophotometric and electrophysiological analysis of the role of the celiac plexus in maintenance of temperature homeostasis during cold stress in rats

The role of the celiac plexus in maintenance of temperature homeostasis in rats exposed to cold stress was studied by histochemical, ultrastructural, and electrophysiological methods. Inhibition of efferent impulsation was found in the preganglionic (splanchnic) nerves and potentiation in postganglionic (superior mesenteric) nerves under the influence of short-term cold stress, leading to a state of mild hypothermia. During cooling of the animals after decentralization of the celiac plexus an increase was observed in the intensity of fluorescence, in the activity of enzymes of energy metabolism, and in hyperplasia of the ultrastructural formations responsible for protein synthesis and the energy supply of the cell. It is suggested that during cold stress, when the flow of efferent impulses along preganglionic nerves is considerably reduced the celiac plexus becomes the center regulating autonomic functions that are involved in the maintenance of temperature homeostasis.Institute of Physiology, Belorussian Academy of Sciences, Minsk. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 659–667, November–December, 1992.     

Delineating the relationship between immune system aging and myogenesis in muscle repair

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.     

Effects of calcitonin on osteoclasts in vivo

Summary Osteoclasts from the tibial metaphyses of young rats treated with porcine calcitonin were studied by electron microscopy. The animals were sacrificed 1 1/2, 4, 8 or 12 hours after injection of the hormone. In survey sections examined by light microscopy the osteoclasts appeared smaller than in control animals. At the ultrastructural level the osteoclasts showed the following alterations: 1) The typical ruffled border was absent. 2) Acid phosphatase was not present in the extracellular space between cell and bone. 3) The number of large vacuoles was decreased and there was no local accumulation of vacuoles in the cytoplasm. 4) The vacuoles did not contain bone crystals. 5) Vacuoles with cell organelles were increased in number. The majority of these vacuoles were identified as autolysosomes because they contained acid phosphatase and the enclosed cell organelles were partially digested. The above changes were present at all time intervals studied.The findings suggest that calcitonin decreases or inhibits bone resorption by osteoclasts. A decreased function of the osteoclasts may contribute to the hypocalcemic effect of the hormone. The increased number of autolysosomes is evidence of an enhanced autophagocytosis. Possible origins of the autolysosomes in osteoclasts are discussed.This research was supported by grants no. 512–819, 512–1545 and 512–1912 from the Danish Medical Research Council. The present observations were first reported at the annual meeting of the Scandinavian Society for Electron Microscopy in Umeå 1973 (Lucht, in press). I wish to thank Professor Arvid B. Maunsbach for valuable discussions and suggestions.     

Proteasome inactivation upon aging and on oxidation-effect of HSP 90

Increases of oxidatively modified protein in the cell have been associated with the aging process. Such an accumulation of damaged protein may be the result of increase in the rate of protein oxidation and/or decrease in the rate of degradation of oxidized protein. The multicatalytic proteinase or proteasome is known to be the major proteolytic system involved in the removal of oxidized protein. We have reported that, after isolation of the 20S proteasome from the liver of young and old male Fischer 344 rat, out of the three peptidase activities (chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolase) we assayed with fluorogenic peptides, the peptidyl-glutamyl peptide hydrolase activity was declining with age to a value approximately 50% of that observed for protease purified from young rats. The proteasome was subjected to metal catalyzed oxidation to determine the susceptibility of the different peptidase activities to oxidative inactivation. Both trypsin-like and peptidyl-glutamyl peptide hydrolase activities were found sensitive to oxidation. Treatment of the proteasome with 4-hydroxy-2-nonenal, a major lipid peroxidation product, was also found to inactivate the trypsin-like activity. However, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation in proteasome preparations contaminated with HSP 90, a protein that often copurifies with the proteasome. Upon addition of HSP 90 to pure 20S active proteasome, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation and from inactivation by treatment with 4-hydroxy-2-nonenal. These results suggest a possible intervention of HSP 90 in response to oxidative stress in preventing the inactivation of the proteasome by oxidative damage. Abbreviations: AAF-amc – Ala-Ala-Phe-7-amido-4-methylcoumarin; LSTR-amc – N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; LLE-na – Leu-Leu-Glu-b-naphthylamide; HSP 90: heat shock protein 90, MCP – multicatalytic proteinase or 20S proteasome.     

The effect of ageing on the SIF-cells in the hypogastric (main pelvic) ganglion of the rat

Summary The effect of ageing on SIF-cells was studied by the formaldehyde-induced fluorescence (FIF) method and by electron microscopy (EM). Microspectrofluorimetry was used to record emission spectra in FIF preparations. In newborn and in young adult (8–12 weeks) rats a single type of SIF-cell emitting greenish-yellow FIF was found while in aged rats a second type of SIF-cell emitting yellowish-brown granular FIF was also present. The intensity of the yellowish-brown FIF was lower than that of the greenish-yellow FIF. Also a few bright cytoplasmic fluorescent areas were occasionally found in some SIF-cells. The distribution of the SIF-cells through the ganglion did not change remarkably with age.In EM after glutaraldehyde-fixation in newborn and in young adult rats two types of small granule-containing (SGC) cells were distinguished according to the size of the dense cored vesicles, 1) 50–150 nm and 2) 50–250 nm. In aged rats, a third type of SGC-cells containing 50×250 nm elongated dense core vesicles could also be distinguished. After KMnO₄-fixation in newborn and in young adult rats the classification was identical with glutaraldehyde-fixation. In aged rats three types of storage granules were found after KMnO₄, 1) 100–300 nm empty vesicles and 2) 100–300 nm vesicles with small dense core, 3) 100–500 nm irregular in shape and filled with electron opaque material with a more dense core.     

Calcium content and concretions of pineal glands of young and old rats

Summary Calcium content and pineal concretions were studied in young (2–3 months) and old (28 months) Wistar rats. Samples, deep-frozen by liquid propane/isopentane and freeze-dried were analysed by means of X-ray microanalysis in a scanning electron microscope. Total semi-quantitative measurements revealed that pineals of old rats showed a marked increase of calcium compared with the pineals of young rats. It is thus suggested that a calcium-rich environment is responsible for the growth of pineal concretions, which only appear in old rats. Pineal calcifications in rats could thus be an indicator of aging and/or of a degenerating state.     

Metabonomic alterations in hippocampus, temporal and prefrontal cortex with age in rats

The metabolic changes in hippocampus, temporal cortex and prefrontal cortex in SD rats along with aging were explored using a metabonomic approach, which based on high resolution “magic angle spinning” ¹H NMR spectroscopy. The metabolite profiles were analyzed by partial least squares-discriminant analysis, and the results showed that the metabolites of the above three brain regions in old rats were dramatically different from that in the adult and young rats. The old rats showed increased myo-inositol and lactate in all of the three brain regions, and decreased N-acetylaspartate in temporal and frontal cortex, Glutamate–GABA level became imbalance in temporal cortex of old rats. In addition, compared with the adult female rats, male rats had higher levels of N-acetylaspartate, taurine, and creatine in temporal or frontal cortex. The age-related metabolic changes may indicate the early functional alterations of neural cells in these brain regions, especially the temporal cortex. The gender-related metabolic changes suggest the significance of the hormonal regulation in brain metabolism. Our work highlights the potential of metabolic profiling to enhance our understanding of biological mechanisms of brain aging.     

Hippocampal levels of dynorphin A (1–8) in neonatal and 16-week-old spontaneously hypertensive rats: Comparisons with DOCA-salt hypertension

In this study the possible role of hippocampal dynorphin in the development of hypertension in spontaneously hypertensive rats (SHR) was investigated by determining dynorphin A (1–8) (DN A (1–8)) levels in hippocampus in 16 week old SRH, Wistar Kyoto (WKY) controls and SHR treated with antihypertensive drugs as well as DOCA-salt hypertensive Sprague Dawley (SD) rats, using radioimmunoassay (RIA). We found that DN A (1–8) was decreased significantly in both dorsal (–68%) and ventral (–58%) hippocampus in SHR compared with WKY rats. Treatment with hydralazine and guanethidine (25 mg/kg/24 hr of each drug in drinking water) for 8 weeks to prevent the development of hypertension in young SHR had no effect on this low hippocampal dynorphin level. We failed to find significant changes in hippocampal DN A (1–8) level in DOCA-salt hypertensive rats. The low level of hippocampal dynorphin existed before the development of hypertension in 6 day neonatal SHR (–73%). Hippocampal Met-enkephalin was unchanged in all experimental groups except for a slight decrease in neonatal SHR. The results establish a genetic difference in the hippocampal dynorphin system of SHR compared with WKY, the significance of which, for the development of hypertension, remains to be investigated.     

Effect of calmodulin on sarcoplasmic reticular Ca2+-transport in the aging heart

Heart failure is common among the elderly and an alteration in myocardial Ca²⁺ transport is believed to be involved in its depressed contractile performance. Although ATP-dependent sarcoplasmic reticular (SR) Ca²⁺ transport has been reported to decrease in old hearts, virtually nothing appears to be known about the Ca²⁺ pump activity of SR in aging myocardium in the presence of calmodulin, one of its endogenous activators. In this study, the activity of the Ca²⁺ pump of aging cardiac SR was assessed in the presence of this endogenous stimulator. This assessment was therefore designed to give additional information about the status of this enzyme in old hearts. Male Sprague-Dawley rats were used and were divided into 3 groups: young (4–6 months old); middle-aged (15–17 months old) and old age (24–25 months old). Purified SR membranes were isolated from ventricular tissues. ATP-dependent Ca²⁺ accumulation by membrane vesicles of middle-aged and old hearts was significantly depressed in comparison to young hearts at all Ca²⁺ concentrations employed in the absence and presence of calmodulin. The activity of this Ca²⁺ transporter was similar in middle-aged and old hearts even in the presence of calmodulin. These results suggest that the activity of the Ca²⁺ pump in SR of aging hearts is depressed even in the presence of calmodulin.C. E. Heyliger is a Scholar of the British Columbia Heart Foundation.     

Non-structural carbohydrate pools in a tropical forest

The pool size of mobile, i.e. non-structural carbohydrates (NSC) in trees reflects the balance between net photosynthetic carbon uptake (source) and irreversible investments in structures or loss of carbon (sink). The seasonal variation of NSC concentration should reflect the sink/source relationship, provided all tissues from root to crown tops are considered. Using the Smithsonian canopy crane in Panama we studied NSC concentrations in a semi-deciduous tropical forest over 22 months. In the 9 most intensively studied species (out of the 17 investigated), we found higher NSC concentrations (starch, glucose, fructose, sucrose) across all species and organs in the dry season than in the wet season (NSC 7.2% vs 5.8% of dry matter in leaves, 8.8/6.0 in branches, 9.7/8.5 in stems, 8.3/6.4 in coarse and 3.9/2.2 in fine roots). Since this increase was due to starch only, we attribute this to drought-constrained growth (photosynthesis less affected by drought than sink activity). Species-specific phenological rhythms (leafing or fruiting) did not overturn these seasonal trends. Most of the stem volume (diameter at breast height around 40 cm) stores NSC. We present the first whole forest estimate of NSC pool size, assuming a 200 t ha^–1 forest biomass: 8% of this i.e. ca. 16 t ha^–1 is NSC, with ca. 13 t ha^–1 in stems and branches, ca. 0.5 and 2.8 t ha^–1 in leaves and roots. Starch alone (ca. 10.5 t ha^–1) accounts for far more C than would be needed to replace the total leaf canopy without additional photosynthesis. NSC never passed through a period of significant depletion. Leaf flushing did not draw heavily upon NSC pools. Overall, the data imply a high carbon supply status of this forest and that growth during the dry season is not carbon limited. Rather, water shortage seems to limit carbon investment (new tissue formation) directly, leaving little leeway for a direct CO₂ fertilization effects.     

Rad5 Template Switch Pathway of DNA Damage Tolerance Determines Synergism between Cisplatin and NSC109268 in Saccharomyces cerevisiae

The success of cisplatin (CP) based therapy is often hindered by acquisition of CP resistance. We isolated NSC109268 as a compound altering cellular sensitivity to DNA damaging agents. Previous investigation revealed an enhancement of CP sensitivity by NSC109268 in wild-type Saccharomyces cerevisiae and CP-sensitive and -resistant cancer cell lines that correlated with a slower S phase traversal. Here, we extended these studies to determine the target pathway(s) of NSC109268 in mediating CP sensitization, using yeast as a model. We reasoned that mutants defective in the relevant target of NSC109268 should be hypersensitive to CP and the sensitization effect by NSC109268 should be absent or strongly reduced. A survey of various yeast deletion mutants converged on the Rad5 pathway of DNA damage tolerance by template switching as the likely target pathway of NSC109268 in mediating cellular sensitization to CP. Additionally, cell cycle delays following CP treatment were not synergistically influenced by NSC109268 in the CP hypersensitive rad5Δ mutant. The involvement of the known inhibitory activities of NSC109268 on 20S proteasome and phosphatases 2Cα and 2A was tested. In the CP hypersensitive ptc2Δptc3Δpph3Δ yeast strain, deficient for 2C and 2A-type phosphatases, cellular sensitization to CP by NSC109268 was greatly reduced. It is therefore suggested that NSC109268 affects CP sensitivity by inhibiting the activity of unknown protein(s) whose dephosphorylation is required for the template switch pathway.     

Accumulation of systematic TPM1 mediates inflammation and neuronal remodeling by phosphorylating PKA and regulating the FABP5/NF‐κB signaling pathway in the retina of aged mice

The molecular mechanisms underlying functional decline during normal brain aging are poorly understood. Here, we identified the actin‐associated protein tropomyosin 1 (TPM1) as a new systemic pro‐aging factor associated with function deficits in normal aging retinas. Heterochronic parabiosis and blood plasma treatment confirmed that systemic factors regulated age‐related inflammatory responses and the ectopic dendritic sprouting of rod bipolar (RBC) and horizontal (HC) cells in the aging retina. Proteomic analysis revealed that TPM1 was a potential systemic molecule underlying structural and functional deficits in the aging retina. Recombinant TPM1 protein administration accelerated the activation of glial cells, the dendritic sprouting of RBCs and HCs and functional decline in the retina of young mice, whereas anti‐TPM1 neutralizing antibody treatment ameliorated age‐related structural and function changes in the retina of aged mice. Old mouse plasma (OMP) induced glial cell activation and the dendritic outgrowth of RBCs and HCs in young mice, and yet TMP1‐depleted OMP failed to reproduce the similar effect in young mice. These results confirmed that TPM1 was a systemic pro‐aging factor. Moreover, we demonstrated that systematic TPM1 was an immune‐related molecule, which elicited endogenous TPM1 expression and inflammation by phosphorylating PKA and regulating FABP5/NF‐κB signaling pathway in normal aging retinas. Interestingly, we observed TPM1 upregulation and the ectopic dendritic sprouting of RBCs and HCs in young mouse models of Alzheimer''s disease, indicating a potential role of TPM1 in age‐related neurodegenerative diseases. Our data indicate that TPM1 could be targeted for combating the aging process.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.