Osteopontin infusion into normal adult rat brain fails to increase cell proliferation in dentate gyrus and subventricular zone.

In the first week after focal ischemia in adult brain, the basal level of neurogenesis increases dramatically in two distinct areas: The dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. It is possible that this remotely induced neurogenesis is the result of a proliferation inducing factor, or factors, diffusing from the infarction to the neurogenic regions. The secreted protein osteopontin (OPN) is a possible factor. In this study, OPN mRNA levels were measured in the cerebral infarction of adult rats that underwent I hour of middle cerebral artery occlusion (MCAO). OPN mRNA levels increased 36.0, 55.0 and 46.7 fold at 6, 24 and 72 hours reperfusion respectively. We also determined whether OPN alone could be responsible for this ischemia-induced neurogenesis. OPN (2.4 microg/day) was infused into the lateral ventricles of the brain in non-ischemic adult male rats, continuously over three days. Bromodeoxyuridine (BrdU) immunohistochemistry was performed and the total BrdU positive (BrdU+) cells were counted. OPN, compared to aCSF infusion, decreased BrdU+ cells in DG and had no significant effect on cell proliferation in the SVZ. This study indicates that osteopontin alone does not increase cell proliferation in the normal adult brain.

Protective effects of glial cell line-derived neurotrophic factor on hippocampal neurons after traumatic brain injury in rats.

OBJECT: The purpose of this study was to evaluate whether glial cell line-derived neurotrophic factor (GDNF) can protect against hippocampal neuronal death after traumatic brain injury (TBI). METHODS: Male Sprague-Dawley rats were subjected to moderate TBI with a controlled cortical impact device while in a state of halothane-induced anesthesia. Then, GDNF or artificial cerebrospinal fluid ([aCSF]; vehicle) was infused into the frontal horn of the left lateral ventricle. In eight brain-injured and eight sham-operated rats, GDNF was infused continuously for 7 days (200 ng/day intracerebroventricularly at a rate of 8.35 ng/0.5 microl/hour). An equal volume of vehicle was infused at the same rate into the remaining eight brain-injured and eight sham-operated rats. Seven days post-injury, all rats were killed. Their brains were sectioned and stained with cresyl violet, and the hippocampal neuronal loss was evaluated in the CA2 and CA3 regions with the aid of microscopy. A parallel set of sections from each brain was subjected to immunoreaction with antibodies against glial fibrillary acidic protein (GFAP; astroglia marker). In the aCSF-treated group, TBI resulted in a significant neuronal loss in the CA2 (60%, p < 0.05) and CA3 regions (68%, p < 0.05) compared with the sham-operated control animals. Compared with control rats infused with aCSF, GDNF infusion significantly decreased the TBI-induced neuronal loss in both the CA2 (58%, p < 0.05) and CA3 regions (51%, p < 0.05). There was no difference in the number of GFAP-positive astroglial cells in the GDNF-infused rats in the TBI and sham-operated groups compared with the respective vehicle-treated groups. CONCLUSIONS: The authors found that GDNF treatment following TBI is neuroprotective.

Spermidine/spermine N1-acetyl transferase activity in rat brain following transient focal cerebral ischemia and reperfusion.

The polyamine system is very sensitive to different pathological states of brain and is perturbed after central nervous system (CNS) injury. Spermidine/Spermine N(1)-acetyl transferase (SSAT) is the key enzyme responsible for interconversion of spermine and spermidine to spermidine and putrescine respectively. In the present study, SSAT activity was evaluated in the rat CNS, following transient focal cerebral ischemia and reperfusion. The middle cerebral artery (MCA) was occluded for 2 h in male spontaneously hypertensive rats by an intraluminal suture technique. Animals were sacrificed at 3-24 h reperfusion following the MCA occlusion and SSAT activity was assayed in cortex and striatum. Results showed that SSAT activity was significantly increased at 12 h reperfusion in cortex and at 9, 12 and 18 h reperfusion in striatum following ischemia compared to sham or contralateral controls. These results demonstrate that polyamine catabolism in the rat CNS is altered following MCA occlusion. In the in vitro ischemia study, SSAT activity was evaluated in primary cortical neuronal cultures at 6-24 h re-oxygenation intervals following oxygen-glucose deprivation for 1 h, and the results from this group show that the enzyme activity increased by about 62% (P<0.05) at 24 h re-oxygenation. This study suggests that the increased SSAT activity may contribute to the increase in putrescine during the post-ischemic period.

31P-MRS-based determination of brain intracellular and interstitial pH: its application to in vivo H+ compartmentation and cellular regulation during hypoxic/ischemic conditions.

In the last decade, significant progress has been made in the characterization of pH regulation in nervous tissue in vitro. However, little work has been directed at understanding how pH regulatory mechanisms function in vivo. We are interested in how ischemic acidosis can effect pH regulation and modulate the extent of post-ischemic brain damage. We used 31P-MRS to determine normal in vivo pH(i) and pH(e) simultaneously in both the isolated canine brain and the intact rat brain. We observed that the 31P(i) peak in the 31P-MRS spectrum is heterogeneous and can be deconvoluted into a number of discrete constituent peaks. In a series of experiments, we identified these peaks as arising from either extracellular or intracellular sources. In particular, we identified the peak representing the neurons and astrocytes and showed that they maintain different basal pH (6.95 and 7.05, respectively) and behave differently during hypoxic/ischemic episodes.

In vivo microdialysis of 2-deoxyglucose 6-phosphate into brain: a novel method for the measurement of interstitial pH using 31P-NMR.

A unique method for simultaneously measuring interstitial (pHe) as well as intracellular (pHi) pH in the brains of lightly anesthetized rats is described. A 4-mm microdialysis probe was inserted acutely into the right frontal lobe in the center of the area sampled by a surface coil tuned for the collection of 31P-NMR spectra. 2-Deoxyglucose 6-phosphate (2-DG-6-P) was microdialyzed into the rat until a single NMR peak was detected in the phosphomonoester region of the 31P spectrum. pHe and pHi values were calculated from the chemical shift of 2-DG-6-P and inorganic phosphate, respectively, relative to the phosphocreatine peak. The average in vivo pHe was 7.24+/-0.01, whereas the average pHi was 7.05+/-0.01 (n = 7). The average pHe value and the average CSF bicarbonate value (23.5+/-0.1 mEq/L) were used to calculate an interstitial Pco2 of 55 mm Hg. Rats were then subjected to a 15-min period of either hypercapnia, by addition of CO2 (2.5, 5, or 10%) to the ventilator gases, or hypocapnia (PCO2 < 30 mm Hg), by increasing the ventilation rate and volume. pHe responded inversely to arterial Pco2 and was well described (r2 = 0.91) by the Henderson-Hasselbalch equation, assuming a pKa for the bicarbonate buffer system of 6.1 and a solubility coefficient for CO2 of 0.031. This confirms the view that the bicarbonate buffer system is dominant in the interstitial space. pHi responded inversely and linearly to arterial PCO2. The intracellular effect was muted as compared with pHe (slope = -0.0025, r2 = 0.60). pHe and pHi values were also monitored during the first 12 min of ischemia produced by cardiac arrest. pHe decreases more rapidly than pHi during the first 5 min of ischemia. After 12 min of ischemia, pHe and pHi values were not significantly different (6.44+/-0.02 and 6.44+/-0.03, respectively). The limitations, advantages, and future uses of the combined microdialysis/31P-NMR method for measurement of pHe and pHi are discussed.