Michelle F. Anderson

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.

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.

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.

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.

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.

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.

Mitochondrial respiratory function and cell death in focal cerebral ischemia.

Abstract : Pyruvate‐supported oxygen uptake was determined as a measure of the functional capacity of mitochondria obtained from rat brain during unilateral middle cerebral artery occlusion and reperfusion. During ischemia, substantial reductions developed in both ADP‐stimulated and uncoupled respiration in tissue from the focus of the affected area in the striatum and cortex. A similar pattern of change but with lesser reductions was seen in the adjacent perifocal tissue. Succinate‐supported respiration was more affected than that with pyruvate in perifocal tissue at 2 h of ischemia, suggesting additional alterations to mitochondrial components in this tissue. Mitochondrial respiratory activity recovered fully in samples from the cortex, but not the striatum, within the first hour of reperfusion following 2 h of ischemia and remained similar to control values at 3 h of reperfusion. In contrast, impairment of the functional capacity of mitochondria from all three regions was seen in the first 3 h of reperfusion following 3 h of ischemia. Extensive infarction generally affecting the cortical focal tissue with more variable involvement of the perifocal tissue developed following 2 h of focal ischemia. Thus, mitochondrial impairment during the first 3 h of reperfusion was apparently not essential for tissue infarction to develop. Nonetheless, the observed mitochondrial changes could contribute to the damage produced by permanent focal ischemia as well as the larger infarcts produced when reperfusion was initiated following 3 h of ischemia.

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.

Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples

The investigation of mitochondrial abnormalities in brain commonly requires isolation of these organelles from small tissue samples. We have modified a mitochondrial isolation procedure based on Percoll density gradient centrifugation to increase the proportion of the total mitochondrial pool recovered while reducing contamination with synaptosomes and related structures containing cytoplasm. Initially, myelin was removed by centrifugation in 12% Percoll in isotonic buffer. The pellet was resuspended, treated with digitonin to break up synaptosomes and similar structures and subjected to discontinuous Percoll density gradient centrifugation. The mitochondrial fraction obtained from this procedure was highly metabolically active and well coupled, exhibiting respiratory control ratios above 5. The recovery of mitochondrial markers using a single rat forebrain as starting material was approximately 18% to 21%. When small tissue samples (approximately 50 mg wet weight) were used as starting material the recovery of the mitochondrial marker was approximately 16%. The ratio of recovery of a mitochondrial marker to the cytoplasmic marker lactate dehydrogenase exceeded 200 in preparations from a single rat forebrain. This is substantially greater than values reported for previously published procedures reflecting both an improved yield of mitochondria and a reduction in cytoplasmic contamination.