Neville W. Knuckey

Choroid Plexus Recovery After Transient Forebrain Ischemia: Role of Growth Factors and Other Repair Mechanisms

Abstract1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus.2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism.3. Between 12 and 24 hr postischemia, there was marked restoration of the Na+, K+, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to “fill in gaps” along the basal lamina caused by necrotic epithelial cell disintegration.4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system.5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms.6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance.7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus–CSF–brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.

The protective effect of hypoxic preconditioning on cortical neuronal cultures is associated with increases in the activity of several antioxidant enzymes

Preconditioning describes a variety of treatments that induce neurons to become more resistant to a subsequent ischemic insult. How preconditioned neurons adapt to subsequent ischemic stress is not fully understood, but is likely to involve multiple protective mechanisms. We hypothesized hypoxic preconditioning induces protection by a coordinated up-regulation of antioxidant enzyme activity. To test this hypothesis, we developed two in vitro models of ischemia/reperfusion, involving oxygen-glucose deprivation (OGD) where neuronal cell death was predominantly by necrosis (necrotic model) or programmed cell death (PCD model). Hypoxic preconditioning 24 h prior to OGD significantly reduced cell death from 83% to 22% in the necrotic model and 68% to 11% in the PCD model. Consistent with the hypothesis, the activity of the antioxidant enzymes glutathione peroxidase, glutathione reductase, and Mn superoxide dismutase were significantly increased by 54%, 73% and 32%, respectively, in neuronal cultures subjected to hypoxic preconditioning. Furthermore, superoxide and hydrogen peroxide concentrations following OGD were significantly lower in the PCD model that had been subjected to hypoxic preconditioning.

In Search of Clinical Neuroprotection After Brain Ischemia. The Case for Mild Hypothermia (35°C) and Magnesium

Background and Purpose— Brain injury after stroke and other cerebral ischemic events is a leading cause of death and disability worldwide. Our purpose here is to argue in favor of combined mild hypothermia (35°C) and magnesium as an acute neuroprotective treatment to minimize ischemic brain injury. Methods and Results— Drawing on our own experimental findings with mild hypothermia and magnesium, and in light of the moderate hypothermia trials in cardiac arrest/resuscitation and magnesium trials in ischemic stroke (IMAGES, FAST-Mag), we bring attention to the advantages of mild hypothermia compared with deeper levels of hypothermia, and highlight the existing evidence for its combination with magnesium to provide an effective, safe, economical, and widely applicable neuroprotective treatment after brain ischemia. With respect to effectiveness, our own laboratory has shown that combined mild hypothermia and magnesium treatment has synergistic neuroprotective effects and reduces brain injury when administered several hours after global and focal cerebral ischemia. Conclusions— Even when delayed, combined treatment with mild hypothermia and magnesium has broad therapeutic potential as a practical neuroprotective strategy. It warrants further experimental investigation and presents a good case for assessment in clinical trials in treating human patients after brain ischemia.

Characterisation of neuronal cell death in acute and delayed in vitro ischemia (oxygen-glucose deprivation) models

Using 96 well microtitre plate sized glass wells we have established and characterised two in vitro ischemia (oxygen-glucose deprivation) models that induce acute or delayed neuronal cell death. In vitro ischemia was induced by washing cortical neuronal cultures with a balanced salt solution either with (acute model) or without (delayed model) 25mM 2-deoxy-d-glucose, and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and adding glucose containing media (delayed model) or removing the balanced salt solution/2-deoxy-d-glucose medium (acute model) and adding glucose containing media. The models were characterised with respect to in vitro ischemia dose duration, cell death time course and for necrosis, apoptosis, autophagy and necroptosis biomarkers. To this end, biomarkers for all four modes of cell death were detected in both in vitro ischemia models, although the time of onset and relative proportion of each cell death mode differed between models. While it is likely that different modes of cell death were activated in the same cell, autophagy appeared to be a prominent cell death mode, especially in the delayed model. Together these models will provide valuable tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput screening system to evaluate potential neuroprotective agents.

Modes of Neuronal Calcium Entry and Homeostasis following Cerebral Ischemia.

One of the major instigators leading to neuronal cell death and brain damage following cerebral ischemia is calcium dysregulation. The neurons inability to maintain calcium homeostasis is believed to be a result of increased calcium influx and impaired calcium extrusion across the plasma membrane. The need to better understand the cellular and biochemical mechanisms of calcium dysregulation contributing to neuronal loss following stroke/cerebral ischemia is essential for the development of new treatments in order to reduce ischemic brain injury. The aim of this paper is to provide a concise overview of the various calcium influx pathways in response to ischemia and how neuronal cells attempts to overcome this calcium overload.

Intravenous administration of magnesium is only neuroprotective following transient global ischemia when present with post-ischemic mild hypothermia

We hypothesized that post-ischemic hypothermia plays an important role in magnesium mediated neuroprotection following global cerebral ischemia. To test this hypothesis, we subjected rats to 8 min of global cerebral ischemia and magnesium treatment with and without post-ischemic body temperature maintenance. In Group 1, rats received an intravenously administered loading dose (LD) of 360 micromol/kg MgSO4 immediately before ischemia followed by a 48-h intravenous infusion (IVI) at either 60, 120 or 240 micromol/kg/h. Animal body temperature was kept at 37+/-0.2 degrees C during ischemia and between 36.6 and 37.8 degrees C for 6 h after ischemia. In Group 2, rats received a 360 micromol/kg MgSO4 LD followed by a 48-h IVI of either 120 or 240 micromol/kg/h MgSO4. In this group, body temperature following ischemia was monitored but not regulated. Control animals in Groups 1 and 2 received normal saline. Seven days after ischemia, hippocampal CA1 neurons were histologically examined. All Group 1 MgSO4-treated and control animals demonstrated less than 6% hippocampal CA1 neuronal survival. In Group 2, the rectal temperature of MgSO4-treated and control animals spontaneously dropped as low as 35.4 degrees C during the 6-h post-ischemia monitoring period. In addition, Group 2 animals that received the LD followed by an IVI of 120 or 240 micromol/kg/h MgSO4 demonstrated 34% (p<0.05) and 20% (p=0.936) CA1 neuronal survival, respectively. The CA1 neuronal survival in saline-treated control animals in both groups was less than 6%. Our data demonstrate only the combination of MgSO4 treatment and post-ischemic mild hypothermia is neuroprotective following global ischemia.

Postischemic intravenous administration of magnesium sulfate inhibits hippocampal CA1 neuronal death after transient global ischemia in rats.

OBJECTIVE We aimed to determine an effective dose schedule for intravenously administered magnesium, to establish its neuroprotective efficacy in both pre- and postischemic treatment paradigms, and to compare the neuroprotective properties of MgSO4 and MgCl2. METHODS Rats that had been subjected to the bilateral carotid artery occlusion plus hypotension model of transient forebrain cerebral ischemia received either an intravenously administered loading dose (LD) of 360 &mgr;mol/kg MgSO4 only or an intravenously administered LD of 360 &mgr;mol/kg followed by a 48-hour intravenous infusion of MgSO4 at either 60, 120, 240, or 480 &mgr;mol/kg/h. For evaluation of the efficacy of MgSO4 after ischemia, the dose (LD, 360 &mgr;mol/kg; infusion, 120 &mgr;mol/kg/h) that provided maximal neuroprotection before ischemia was administered 4, 8, 12, or 24 hours after ischemia. MgCl2 (LD, 360 &mgr;mol/kg; infusion, 120 &mgr;mol/kg/h) was administered before and 8 hours after ischemia. At 7 days after ischemia, hippocampal CA1 neurons were histologically examined for protection. RESULTS Animals that received the LD only demonstrated 33% hippocampal CA1 neuronal survival. Animals that received the LD followed by continuous infusion of MgSO4 at either 60, 120, 240, or 480 &mgr;mol/kg/h demonstrated 30, 80, 16, and less than 5% CA1 neuronal survival, respectively. MgSO4 treatment commencing at 4, 8, 12, or 24 hours resulted in 82, 71, 52, and 33% CA1 neuronal survival, respectively. Preischemic and 8-hour postischemic administration of MgCl2 resulted in 50% and less than 5% CA1 neuronal survival, respectively. CONCLUSION These results demonstrate a neuroprotective intravenous dose of MgSO4, which is effective when administered before or late after ischemia, and a previously uncharacterized dose-response curve for MgSO4.

Combined magnesium and mild hypothermia (35 °C) treatment reduces infarct volumes after permanent middle cerebral artery occlusion in the rat at 2 and 4, but not 6 h

BACKGROUND AND PURPOSE Using transient focal and global cerebral ischemia models in the rat, we have previously shown that MgSO4 is not neuroprotective unless it is combined with mild hypothermia. This study establishes a therapeutic time window for combined MgSO4 and mild hypothermia treatment after permanent middle cerebral artery occlusion (MCAO). METHODS Rats were subjected to permanent intraluminal thread MCAO and animals were treated 2, 4 or 6 h after ischemia with a MgSO4 infusion (360 micromol/kg, then 120 micromol/kg/h) and mild hypothermia (35 degrees C) or with vehicle for 24 h. At the 2 h time point, treatment with hypothermia alone and MgSO4 alone were also assessed. Infarct volumes were measured 48 h after MCAO induction. RESULTS After permanent MCAO, combined MgSO4 and hypothermia treatment reduced infarct volumes by 54% at 2 h (P = 0.048) and by 39% at 4 h (P = 0.012), but there was no treatment effect detected at 6 h or in the hypothermia alone or MgSO4 alone groups. CONCLUSIONS These findings support our earlier work highlighting the neuroprotective effect of MgSO4 when combined with mild hypothermia, even when treatment is delayed by several hours.

Establishment of neuronal in vitro models of ischemia in 96-well microtiter strip-plates that result in acute, progressive and delayed neuronal death

Using 96-well microtiter strip-plates we established in vitro ischemia models with acute, progressive and delayed neuronal death onset. In vitro ischemia was induced by washing neuronal cultures with a balanced salt solution with (acute/delayed models) or without (progressive model) 25 mM 2-deoxy-D-glucose and incubating in an anaerobic chamber. Reperfusion was performed by removing cultures from the anaerobic chamber and washing and/or adding Dulbeccos modified Eagle medium containing N2 supplement. Acute neuronal death resulted in cell swelling during in vitro ischemic incubation with the majority of neurons appearing swollen and necrotic within 3 h post-insult. Progressive neuronal death was characterized by cell shrinkage during and immediately following in vitro ischemia with increasing neuronal degeneration resembling both necrosis and apoptosis over a 24-h period post-in vitro ischemia. Delayed neuronal death was induced by glutamate-receptor blockade during in vitro ischemia. Neurons appeared morphologically normal immediately following and up to 6 h after in vitro ischemia and then started to degenerate over the next 42 h by a process resembling apoptosis. We monitored oxygen consumption during in vitro ischemia and found it to be similar for the three models and have shown that plastic culture wells store oxygen. The establishment of acute, progressive and delayed in vitro models of ischemia using 96-well microtiter strip-plates will provide useful tools to further investigate ischemic neuronal death/survival mechanisms and provide a high-throughput system to evaluate potential neuroprotective agents. Oxygen storage in plastic culture wells is likely to contribute to the extended oxygen- and oxygen-glucose-deprivation times required to induce significant neuronal injury in vitro.

Erythropoietin preconditioning in neuronal cultures : Signaling, protection from in vitro ischemia, and proteomic analysis

In this study we confirmed the presence of the erythropoietin (EPO) receptor on both cultured cortical neurons and PC12 cells and showed that EPO can induce changes in p38, ERK, and JNK signaling molecules in these cells. We induced EPO preconditioning in cortical neuronal cultures that protected neurons from a subsequent in vitro ischemic insult (transient oxygen‐glucose deprivation). To investigate downstream changes in protein expression in EPO‐preconditioned cortical neuronal cultures, we used two‐dimensional gel electrophoresis. Overall, EPO preconditioning resulted in protein up‐regulation, and, from 84 of the most differentially expressed proteins selected for identification, the proteins or tentative proteins were identified in 57 cases, representing 40 different proteins. Different protein spots representing the same or closely related protein(s) occurred for 13 of the identified proteins and are likely to represent posttranslational modifications or proteolytic fragments of the protein. Two proteins (78‐kD glucose‐regulated protein and tropomyosin, fibroblast isoform 1) were detected in control neuronal cultures, but not following EPO preconditioning treatment, whereas one protein (40S ribosomal protein SA) was detected only following EPO preconditioning. Most of the other proteins identified had not previously been associated with EPO preconditioning and will aid in the understanding of EPOs neuroprotective response and possibly the development of new therapeutic interventions to inhibit neuronal death in acute and chronic neurodegenerative diseases.