Trauma-induced reactive gliosis is reduced after treatment with octanol and carbenoxolone.

BACKGROUND: Reactive gliosis and scar formation after brain injury can inhibit the recovery process. As many glial cells utilize gap junctions for intercellular signaling, this study investigated whether two commonly used gap junction blockers, octanol and carbenoxolone, could attenuate reactive gliosis following a minor traumatic brain injury. METHODS: Octanol (710 mg/kg) or carbenoxolone (90 mg/kg) was administered 30 minutes before or after a needle track injury in adult male Sprague-Dawley rats. To mark dividing cells, animals were injected with bromodeoxyuridine (BrdU; 150 mg/kg) intraperitoneally two times per day, 8 hours apart and killed 2 days later. Immunohistochemistry for BrdU and markers for reactive glial cells [glial fibrillary acidic protein (GFAP), ED1, and NG2] were investigated using immunohistochemistry and western blot techniques. RESULTS: Two days after injury, increased cellular proliferation, activated astrocytes and microglia, and upregulation of NG2 expression were observed surrounding the injury site. Octanol and carbenoxolone administrated prior to injury significantly decreased cell proliferation by 60 and 70% respectively. The distance of GFAP immunoreactive astrocytes from the wound margin was decreased by 32 and 18% when octanol was administrated prior to or post injury respectively. Treatment with octanol also decreased the number of reactive microglia by 55% and, when administrated prior to injury, octanol reduced the distance of NG2 expression from the wound by 48%. CONCLUSION: The present study demonstrates that two important components of reactive gliosis, cellular activation and proliferation, can be attenuated by octanol and carbenoxolone.

Repeated transient sulforaphane stimulation in astrocytes leads to prolonged Nrf2-mediated gene expression and protection from superoxide-induced damage.

Oxidative stress is a major contributor to slowly developing diseases like Parkinson's disease, Alzheimer's disease and cancer and one of the main causes of tissue damage following ischemic insults in the brain. Nrf2 is a transcription factor responsible for much of the inducible cellular defense against oxidative stress. Nrf2 can also be activated by xenobiotics like sulforaphane, a component highly enriched in cruciferous vegetables such as broccoli. Ingestion of broccoli or sulforaphane results in long-term protection against radical damage, although absorbed sulforaphane is cleared from the body within a few hours. Here we have examined whether the prolonged protection induced by sulforaphane is explained by a slow down regulation of the Nrf2 response. Furthermore, to simulate daily ingestion of sulforaphane, we examined the hypothesis that repeated transient sulforaphane stimulation results in an accumulation of Nrf2-mediated gene expression and an increased protection against oxidative damage. The kinetics of sulforaphane-induced Nrf2 response was studied in astrocytes, a cell type known to be highly involved in the defense against oxidative stress in the brain. Sulforaphane stimulation for 4 h induced an Nrf2-dependent increase of Nqo1 and Hmox1 mRNA that remained elevated for 24 h, and the corresponding proteins remained elevated for over 48 h. In addition, peroxide-clearing activity and the levels of glutathione were elevated for more than 20 h after stimulation for 4 h with sulforaphane, resulting in an increased resistance to superoxide-induced cell damage. Repeated sulforaphane stimulation resulted in an accumulation of mRNA and protein levels of Nqo1 and a persistent cell protection against oxidative damage. These findings indicate that brief stimulation of the Nrf2 pathway by sulforaphane results in long-lasting elevation of endogenous antioxidants in astrocytes. The findings also demonstrate that part of this response can be built up by repeated transient stimulation, possibly explaining how intermittent intake of sulforaphane can result in long-term protection from radical-induced disease.

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation.

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.

Peripheral infusion of insulin-like growth factor-I increases the number of newborn oligodendrocytes in the cerebral cortex of adult hypophysectomized rats.

We have previously shown that recombinant human (rh) IGF-I induces cell proliferation and neurogenesis in the hippocampus of hypophysectomized rats. In the current investigation, we determined the effects of rhIGF-I on proliferation and differentiation in the cerebral cortex. Adult hypophysectomized rats were injected with bromodeoxyuridine (BrdU) to label newborn cells (once a day for the first 5 d), and rhIGF-I was administered peripherally for 6 or 20 d. In the cerebral cortex, the number of BrdU-labeled cells increased after 20 d but not after 6 d of rhIGF-I infusion. This suggests that rhIGF-I enhances the survival of newborn cells in the cerebral cortex. Using BrdU labeling combined with the oligodendrocyte-specific markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase, we demonstrated an increase in oligodendrogenesis in the cerebral cortex. The total amount of myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase was also increased on Western blots of homogenates of the cerebral cortex, confirming the immunohistochemical findings. Also, we observed an increase in the number of capillary-associated BrdU-positive cells, although total capillary area was not increased. rhIGF-I treatment did not affect cortical astrogliogenesis and neurogenesis was not observed. The ability of rhIGF-I to induce cortical oligodendrogenesis may have implications for the regenerative potential of the cortex.

Immunohistochemical distribution of glucose-dependent insulinotropic polypeptide in the adult rat brain.

We have previously demonstrated that glucose-dependent insulinotropic polypeptide (GIP; gastric inhibitory polypeptide) is present in the adult rat hippocampus. This finding leads to the conclusion that all members of the secretin-glucagon family of gastrointestinal regulatory polypeptides can be found in the brain. To investigate the localization of GIP-producing cells, we used immunohistochemistry on sections of the adult rat brain. High levels of GIP immunoreactivity were observed in the olfactory bulb, hippocampus, and Purkinje cells in the cerebellum. Moreover, a moderate but distinct GIP immunoreactivity was observed in the cerebral cortex, amygdala, substantia nigra, and lateral septal nucleus as well as in several nuclei in the thalamus, hypothalamus, and brainstem. GIP immunoreactivity was frequently found to colocalize with the neuronal marker NeuN but never with the glial marker glial fibrillary acidic protein. Thus, GIP appears to be mainly neuronal to its distribution. This widespread distribution of GIP-immunoreactive cells suggests the involvement of GIP in various neuronal functions and suggests that GIP may act as a neurotransmitter or neuromodulator. This is the first characterization of the anatomical distribution of GIP-immunoreactive cells in the rat brain providing an anatomical framework for future investigations regarding the functions of GIP in the central nervous system.

Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension.

The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone cells reach the olfactory bulb (OB) in rodents. However, the RMS in the adult human brain has been elusive. We demonstrate the presence of a human RMS, which is unexpectedly organized around a lateral ventricular extension reaching the OB, and illustrate the neuroblasts in it. The RMS ensheathing the lateral olfactory ventricular extension, as seen by magnetic resonance imaging, cell-specific markers, and electron microscopy, contains progenitor cells with migratory characteristics and cells that incorporate 5-bromo-2'-deoxyuridine and become mature neurons in the OB.

Glucose-dependent insulinotropic polypeptide is expressed in adult hippocampus and induces progenitor cell proliferation.

The hippocampal dentate gyrus (DG) is an area of active proliferation and neurogenesis within the adult brain. The molecular events controlling adult cell genesis in the hippocampus essentially remain unknown. It has been reported previously that adult male and female rats from the strains Sprague Dawley (SD) and spontaneously hypertensive (SHR) have a marked difference in proliferation rates of cells in the hippocampal DG. To exploit this natural variability and identify potential regulators of cell genesis in the hippocampus, hippocampal gene expression from male SHR as well as male and female SD rats was analyzed using a cDNA array strategy. Hippocampal expression of the gene-encoding glucose-dependent insulinotropic polypeptide (GIP) varied strongly in parallel with cell-proliferation rates in the adult rat DG. Moreover, robust GIP immunoreactivity could be detected in the DG. The GIP receptor is expressed by cultured adult hippocampal progenitors and throughout the granule cell layer of the DG, including progenitor cells. Thus, these cells have the ability to respond to GIP. Indeed, exogenously delivered GIP induced proliferation of adult-derived hippocampal progenitors in vivo as well as in vitro, and adult GIP receptor knock-out mice exhibit a significantly lower number of newborn cells in the hippocampal DG compared with wild-type mice. This investigation demonstrates the presence of GIP in the brain for the first time and provides evidence for a regulatory function for GIP in progenitor cell proliferation.

Hypoxic preconditioning confers long-term reduction of brain injury and improvement of neurological ability in immature rats.

Exposure to preconditioning (PC) hypoxia 24 h before a severe hypoxic-ischemic (HI) insult reduces development of injury in the immature brain. Several protective regimens have proved effective in the short-term but not in the long-term perspective. The aim of the present study, therefore, was to evaluate the PC effect on long-term morphologic and neurologic outcome in the developing brain. Six-day-old rats were subjected to hypoxia (36 degrees C, 8.0% O2; PC/HI group) and sham controls to normoxia (36 degrees C; HI group) for 3 h. Twenty-four hours later, all rats were exposed to cerebral HI produced by unilateral carotid artery occlusion combined with 1 h, 15 min of hypoxia (36 degrees C, 7.7% O2). A cylinder test was used to evaluate forelimb asymmetry to determine sensorimotor function at 4, 6, and 8 wk of age. Spatial/cognitive ability was assessed by Morris water maze trials at 7 wk of recovery. Neuropathologic analysis was performed 8 wk after insult. Brain damage was reduced (p<0.0001) in PC/HI (45.0+/-11.1 mm3) in comparison with HI (159.3+/-12.2 mm3) rats. A bias for using the ipsilateral forelimb in wall movements was observed in the cylinder test in HI compared with PC/HI rats at 4 (p<0.001), 6 (p<0.01), and 8 (p<0.0001) wk of age. Results of the Morris water maze test revealed differences (p<0.0001) in average path length between groups on the third and fourth day of trials. Hypoxic PC before HI reduced brain injury by 72% at 8 wk after the insult and provided long-term improvement of sensorimotor and spatial/cognitive functions.

Glutathione monoethyl ester provides neuroprotection in a rat model of stroke.

Oxidative stress plays an important role in the development of tissue damage following transient focal cerebral ischaemia. Glutathione is a central component in the antioxidant defence of cells. We have previously shown a close association between mitochondrial glutathione loss and cell death following middle cerebral artery (MCA) occlusion. Glutathione monoethyl ester increases cellular glutathione and is particularly effective in increasing the mitochondrial pool. In the present investigation, we infused glutathione monoethyl ester into the third ventricle during 2 h of MCA occlusion and 48 h of reperfusion. Infarct size was reduced from 46% of the total ischaemic hemisphere in saline-treated animals to 16% following ester treatment. Thus, glutathione monoethyl ester provides neuroprotection following transient focal cerebral ischaemia.

Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia.

Glutathione is a central component in the antioxidant defences of cells. We have recently reported an early and selective loss of total (reduced plus oxidised) glutathione from mitochondria isolated from rat brain following occlusion of the middle cerebral artery. This mitochondrial glutathione depletion showed an apparent association with the tissue damage that developed during subsequent reperfusion, suggesting that it could be an important determinant of susceptibility to cell loss. In the present study, we have investigated whether in vivo treatment with glutathione ethyl ester can modulate mitochondrial glutathione in the brain and whether this treatment can influence the response to focal ischemia. In further support of our previous findings, middle cerebral artery occlusion caused a duration-dependent partial loss of mitochondrial glutathione. Bilateral injections of glutathione ethyl ester immediately prior to induction of unilateral focal ischemia resulted in a substantial increase in glutathione in mitochondria from the striatum of both the non-ischemic hemisphere (190% of saline-treated controls) and the ischemic hemisphere (240% of controls) at 2h after arterial occlusion. Total tissue glutathione was not affected by the ester treatment at this time. A smaller increase in mitochondrial glutathione was observed at 3h of occlusion in the non-ischemic striatum following ester treatment but at this time point glutathione was not significantly altered in mitochondria from the ischemic hemisphere. Pre-ischemic treatment with glutathione ester did not significantly change the volume of tissue infarction assessed at 48 h following ischemia for 2 or 3h. These studies demonstrate that glutathione ethyl ester is a highly effective modulator of the mitochondrial glutathione pool in the intact brain and provides a useful means for further investigating the role of this antioxidant in the development of tissue damage in ischemia and other brain disorders.

Astrocytes and stroke: networking for survival?

Astrocytes are now known to be involved in the most integrated functions of the central nervous system. These functions are not only necessary for the normally working brain but are also critically involved in many pathological conditions, including stroke. Astrocytes may contribute to damage by propagating spreading depression or by sending proapoptotic signals to otherwise healthy tissue via gap junction channels. Astrocytes may also inhibit regeneration by participating in formation of the glial scar. On the other hand, astrocytes are important in neuronal antioxidant defense and secrete growth factors, which probably provide neuroprotection in the acute phase, as well as promoting neurogenesis and regeneration in the chronic phase after injury. A detailed understanding of the astrocytic response, as well as the timing and location of the changes, is necessary to develop effective treatment strategies for stroke patients.

The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions.

Glutathione is a key cellular antioxidant that is contained in both cytoplasmic and mitochondrial compartments. Previous investigations indicate that depletion of the mitochondrial pool of glutathione can greatly reduce cell viability. In the present investigation, the effect of focal cerebral ischemia on total (reduced plus oxidized) glutathione in mitochondria was assessed using a rat model of middle cerebral artery occlusion. Total glutathione was substantially decreased in mitochondria prepared from severely ischemic focal tissue in both the cerebral cortex and striatum at 2 h of vessel occlusion and persisted for at least the first 3 h of reperfusion. The loss of mitochondrial glutathione was not associated with decreases of the total tissue glutathione content and was not due to the formation of mixed disulfides with mitochondrial proteins. Thus, an imbalance between uptake and release from the mitochondria in the ischemic tissue provides the most likely explanation for the loss. Decreases in glutathione also developed in mitochondria from the moderately ischemic perifocal tissue when the period of arterial occlusion was extended to 3 h. The presence of mitochondrial glutathione depletion during ischemia showed an apparent close association with the subsequent development of tissue infarction. These findings are consistent with a role for the glutathione depletion in determining the susceptibility of brain tissue to focal ischemia.

Insulin-like growth factor-I and neurogenesis in the adult mammalian brain.

In most brain regions of highly developed mammals, the majority of neurogenesis is terminated soon after birth. However, new neurons are continually generated throughout life in the subventricular zone and the dentate gyrus of the hippocampus. Insulin-like growth factor-I (IGF-I) is a polypeptide hormone that has demonstrated effects on these progenitor cells. IGF-I induces proliferation of isolated progenitors in culture, as well as affecting various aspects of neuronal induction and maturation. Moreover, systemic infusion of IGF-I increases both proliferation and neurogenesis in the adult rat hippocampus, and uptake of serum IGF-I by the brain parenchyma mediates the increase in neurogenesis induced by exercise. Neurogenesis in the adult brain is regulated by many factors including aging, chronic stress, depression and brain injury. Aging is associated with reductions in both hippocampal neurogenesis and IGF-I levels, and administration of IGF-I to old rats increases neurogenesis and reverses cognitive impairments. Similarly, stress and depression also inhibit neurogenesis, possibly via the associated reductions in serotonin or increases in circulating glucocorticoids. As both of these changes have the potential to down regulate IGF-I production by neural cells, stress may inhibit neurogenesis indirectly via downregulation of IGF-I. In contrast, brain injury stimulates neurogenesis, and is associated with upregulation of IGF-I in the brain. Thus, there is a tight correlation between IGF-I and neurogenesis in the adult brain under different conditions. Further studies are needed to clarify whether IGF-I does indeed mediate neurogenesis in these situations.

Mitochondrial contributions to tissue damage in stroke.

Tissue infarction, involving death of essentially all cells within a part of the brain, is a common pathology resulting from stroke and an important determinant of the long-term consequences of this disorder. The cell death that leads to infarct formation is likely to be the result of multiple interacting pathological processes. A range of factors, including the severity of the ischemic insult and whether this is permanent or reversed, determine which mechanisms predominate. Although evaluating mitochondrial properties in intact brain is difficult, evidence for several potentially deleterious responses to cerebral ischemia or post-ischemic reperfusion have been obtained from investigations using animal models of stroke. Marked changes in ATP and related energy metabolites develop quickly in response to occlusion of a cerebral artery, as expected from limitations in the delivery of oxygen and glucose. However, these alterations are often only partially reversed on reperfusion despite improved substrate delivery. Ischemia-induced decreases in the mitochondrial capacity for respiratory activity probably contribute to the ongoing impairment of energy metabolism during reperfusion and possibly also to the magnitude of changes seen during ischemia. Conditions during reperfusion are likely to be conducive to the induction of the permeability transition in mitochondria. There are as yet no well-characterized techniques to identify this change in the intact brain. However, the protective effects of some agents that block formation of the transition pore are consistent with both the induction of the permeability transition during early recirculation and a role for this in the development of tissue damage. Release of cytochrome c into the cytoplasm of cells has been observed with both permanent and reversed ischemia and could trigger the death of some cells by apoptosis, a process which probably contributes to the expansion of the ischemic lesion. Mitochondria are also likely to contribute to the widely-accepted role of nitric oxide in the development of ischemic damage. These organelles are a probable target for the deleterious effects of this substance and can also act as a source of superoxide for reaction with the nitric oxide to produce the damaging species, peroxynitrite. Further characterization of these mitochondrial responses should help to elucidate the mechanisms of cell death due to cerebral ischemia and possibly point to novel sites for therapeutic interventions in stroke.