Ischemia: astrocytes show their sensitive side.
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Biomedical subjects
R Fern
Publications and source records attributed to R Fern.
Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter.
We tested the hypothesis that astrocytic glycogen sustains axon function during and enhances axon survival after 60 min of glucose deprivation. Axon function in the rat optic nerve (RON), a CNS white matter tract, was monitored by measuring the area of the stimulus-evoked compound action potential (CAP). Switching to glucose-free artificial CSF (aCSF) had no effect on the CAP area for approximately 30 min, after which the CAP rapidly failed. Exposure to glucose-free aCSF for 60 min caused irreversible injury, which was measured as incomplete recovery of the CAP. Glycogen content of the RON fell to a low stable level 30 min after glucose withdrawal, compatible with rapid use in the absence of glucose. An increase of glycogen content induced by high-glucose pretreatment increased the latency to CAP failure and improved CAP recovery. Conversely, a decrease of glycogen content induced by norepinephrine pretreatment decreased the latency to CAP failure and reduced CAP recovery. To determine whether lactate represented the fuel derived from glycogen and shuttled to axons, we used the lactate transport blockers quercetin, alpha-cyano-4-hydroxycinnamic acid (4-CIN), and p-chloromercuribenzene sulfonic acid (pCMBS). All transport blockers, when applied during glucose withdrawal, decreased latency to CAP failure and decreased CAP recovery. The inhibitors 4-CIN and pCMBS, but not quercetin, blocked lactate uptake by axons. These results indicated that, in the absence of glucose, astrocytic glycogen was broken down to lactate, which was transferred to axons for fuel.
4-Chloromercuribenzenesulfonate↗
Rapid ischemic cell death in immature oligodendrocytes: a fatal glutamate release feedback loop.
Ischemic injury of immature oligodendrocytes is a major component of the brain injury associated with cerebral palsy, the most common human birth disorder. We now report that cultured immature oligodendrocytes [O4(+)/galactoceramide (GC)(-)] are exquisitely sensitive to ischemic injury (80% of cells were dead after 25.5 min of oxygen and glucose withdrawal). This rapid ischemic cell death was mediated by Ca(2+) influx via non-NMDA glutamate receptors. The receptors were gated by the release of glutamate from the immature oligodendrocytes themselves via reverse glutamate transport and included a significant element of autologous feedback of glutamate from cells onto their own receptors. High (> or = 100 microM) extracellular glutamate was protective against ischemic injury as a result of non-NMDA glutamate receptor desensitization. Other potential pathways of Ca(2+) influx, such as voltage-gated Ca(2+) channels, NMDA receptors, or the Na(+)-Ca(2+) exchanger, did not significantly contribute to ischemic Ca(2+) influx or cell injury. Release of Ca(2+) from intracellular stores was also not an important factor. In agreement with previous studies, more mature oligodendrocytes (O4(-)/GC(+)) were found to be less sensitive to ischemic injury than were the immature cells studied here.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Intracellular calcium and cell death during ischemia in neonatal rat white matter astrocytes in situ.
The major pathological correlate of cerebral palsy is ischemic injury of CNS white matter. Histological studies show early injury of glial cells and axons. To investigate glial cell injury, I monitored intracellular Ca2+ and cell viability in fura-2-loaded neonatal rat white matter glial cells during ischemia. Fura-2 fixation combined with immunohistochemistry revealed that fura-2-loaded cells were GFAP+/O4(-) and were therefore a population of neonatal white matter astrocytes. Significant ischemic Ca2+ influx was found, mediated by both L- and T-type voltage-gated Ca2+ channels. Ca2+ influx via T-type channels was the most important factor during the initial stage of ischemia and was associated with significant cell death within 10-20 min of the onset of ischemia. The Na+-Ca2+ exchanger acted to remove cytoplasmic Ca2+ throughout the ischemic and recovery periods. Neither the release of Ca2+ from intracellular stores nor influx via glutamate-gated channels contributed to the rise in intracellular Ca2+ during ischemia. Ischemic cell death was reduced significantly by removing extracellular Ca2+ or by blocking voltage-gated Ca2+ channels. The exclusively voltage-gated Ca2+ channel nature of the Ca2+ influx, the role played by T-type Ca2+ channels, the protective effect of the Na+-Ca2+ exchanger, and the lack of significant Ca2+ release from intracellular stores are features of ischemia that have not been reported in other CNS cell types.
Animals↗
Changes in [Ca2+]0 during anoxia in CNS white matter.
Irreversible anoxic injury of axons in the rat optic nerve requires the presence of extracellular Ca2+. To test the hypothesis that Ca2+ enters an intracellular compartment during anoxia we monitored [Ca2+]0 in this CNS white matter tract using ion-sensitive microelectrodes. Periods of anoxia lasting 15 min resulted in a rapid, reversible increase in [Ca2+]0 accompanied by transient loss of nerve conduction. This increase in [Ca2+]0 was apparently the result of extracellular space shrinkage. Anoxic periods lasting 60 min resulted in an initial rise followed by a sustained fall in [Ca2+]0, indicative of net influx of Ca2+ into an intracellular compartment. Following reoxygenation after 60 min of anoxia, [Ca2+]0 slowly returned toward control levels but nerve conduction recovered incompletely, indicating irreversible loss of function. Removal of bath Ca2+ lowered [Ca2+]0 to about 100 microM, prevented the anoxia-induced fall in [Ca2+]0, and protected against irreversible loss of the compound action potential.
Animals↗
Axon conduction and survival in CNS white matter during energy deprivation: a developmental study.
We investigated the postnatal development of axon sensitivity to the withdrawal of oxygen, glucose, or the combined withdrawal of oxygen + glucose in the isolated rat optic nerve (a CNS white matter tract). Removal of either oxygen or glucose for 60 min resulted in irreversible injury in optic nerves from adult rats, assessed by loss of the evoked compound action potential (CAP). Optic nerves at ages 45 min caused the selective loss of late CAP components; this was not seen with oxygen deprivation. The amplitude of the early component recovered to 94.8% of control after 60 min of glucose withdrawal, although total CAP area recovered to only 42.3%. Combined oxygen + glucose withdrawal for 60 min produced a greater degree of permanent CAP loss than 60 min of glucose or oxygen withdrawal individually in optic nerves from rats older than P4. Younger than P4 optic nerves showed no permanent loss of function from 60 min of combined oxygen + glucose withdrawal. Unexpectedly, optic nerves from P21-P49 rats recovered significantly less after all three conditions than adult opticnerves (>P50). It is probable that this period of final myelination corresponds to a time of heightened metabolic activity in white matter. The tolerance of CNS white matter to energy deprivation can be categorized into four stages that are correlated with specific developmental features: premyelination (P0-P4), highly tolerant to anoxia, aglycemia and combined anoxia/aglycemia; early myelination (P5-P20), partially tolerant of anoxia and aglycemia but not to combined anoxia/aglycemia; late myelination (P21-P49), very low tolerance of anoxia, aglycemia and combined anoxia/aglycemia; and mature (>P50), low tolerance of anoxia, aglycemia and combined anoxia/aglycemia. The relative resistance of optic nerve function to glucose withdrawal in the presence of oxygen, compared with glucose withdrawal in the absence of oxygen, is presumably due to the presence of oxygen-dependent energy reserves such as astrocytic glycogen, amino acids. and phospholipids.
Action Potentials↗
Ischemic injury of optic nerve axons: the nuts and bolts.
Anterior ischemic optic neuropathy is the most common cause of persistent monocular visual loss in persons over the age of 50. At the heart of this form of optic neuropathy is a sequence of cytoplasmic and membrane events that culminate in axonal destruction. Early depletion of ATP is followed by membrane depolarization, influx of Na+ and Ca2+ via specific voltage-gated channels and reverse operation of the Na+/Ca2+ exchange protein. Toxic Ca2+ overload is the ultimate consequence of these events. Preventing or modulating any of these well-defined steps mitigates against the development of anoxic injury. Translating these molecular insights about how optic nerve axons are damaged by ischemia-like conditions into clinical gains remains the challenge for the future.
Adenosine Triphosphate↗
Does astrocytic glycogen benefit axon function and survival in CNS white matter during glucose deprivation?
Axons, the functional elements in CNS white matter, are frequently injured by ischemia, especially in the context of stroke. The pathophysiology of axonal injury induced by energy deprivation has been analyzed in the rat optic nerve and involves excessive calcium influx by way of reverse Na+/Ca2+ exchange and Ca2+ channels. Evidence is presented that CNS axonal function can be supported in the absence of glucose by intrinsic energy reserves provided through the breakdown of astrocytic glycogen. It is argued that energy is transferred from astrocytes to axons in the form of lactate, which is able to maintain axonal function when substituted for glucose. These observations complement the increasingly convincing hypothesis that astrocytes and neurons interact metabolically, both in the course of normal activity and under pathological conditions such as ischemia. The emerging picture would be no surprise to Camillo Golgi, who predicted a close facsimile of this glial-neuronal interaction more than a century ago.
Animals↗
Evidence for functional co-activation of N-methyl-D-aspartate receptors by glycine.
The excitatory neurotransmitter glutamate acts at several receptor subtypes in the CNS, including N-methyl-D-aspartate (NMDA) receptors. The inhibitory neurotransmitter glycine is a co-agonist of NMDA receptors and functional glycinergic co-activation of NMDA receptors is theoretically possible due to the presence of background glutamate. We report the first high fidelity recording of a distinct NMDA receptor excitatory post-synaptic potential (EPSP) in the intact mammalian CNS. The EPSP was evoked by multi-shock activation of a glycinergic pathway rather than a glutamatergic pathway and had characteristics most compatible with glycine, rather than glutamate, activation of NMDA receptors.
Animals↗
Autoprotective mechanisms in the CNS: some new lessons from white matter.
Anoxia/ischemia in the CNS is a common and devastating phenomenon. It is possible that the best hopes for protection against anoxic/ischemic injury may involve recruiting and/or augmenting any autoprotective systems that evolution has provided for the CNS. We describe here the existence of such an autoprotective system present in CNS white matter. White matter is both well suited to studying extrasynaptic systems, such as the system we describe here, and is a highly appropriate target for research into anoxic-ischemic injury in its own right. We show that white matter contains functional GABAB and adenosine receptors that respond to an anoxic efflux of GABA and adenosine by recruiting a convergent intracellular mechanism involving protein kinase C (PKC). The net result of this receptor-mediated cascade is an increase in resistance to anoxia, which presumably allows CNS white matter to tolerate better a common class of ischemic events that are located solely in white matter and that comprises approximately 25% of all strokes seen clinically.
Animals↗
Cd(2+)-induced injury in CNS white matter.
1. The affect of extracellular Cd2+ on CNS white matter was studied using an isolated rat optic nerve preparation. A 100-min exposure to 200 microM Cd2+ reduced the area of the compound action potential (CAP) recorded from the optic nerve to 32.6 +/- 3.8% (mean +/- SE) of the preexposure area, compared with a reduction to 74.9 +/- 2.9% after 100 min in control conditions (P > 0.001). This CAP reduction was not reversed after 120 min of reperfusion with Cd(2+)-free solution, or by perfusion with Cd2+ chelators. 2. Cd(2+)-induced CAP loss occurred in the absence of extracellular Ca2+. Increasing extracellular Ca2+ concentration to 16 mM, however, prevented Cd(2+)-induced CAP loss. Once evident, Cd(2+)-induced CAP reduction could not subsequently be reversed by addition of 16 mM Ca2+. 3. Low concentrations of Cd2+ (60 microM) did not significantly reduce CAP area. This concentration of Cd2+ combined with high extracellular K+ (30 mM) caused CAP loss that was blocked by 10 microM nifedipine, an antagonist of L-type voltage-gated Ca2+ channels. 4. Treatment with pharmacological inhibitors of membrane proteins known to be inhibited by Cd2+ did not affect the CAP. These included inhibitors of voltage-gated Ca2+ channels, Ca(2+)-activated K+ channels, Ca(2+)-ATPase and the Na+/Ca2+ exchanger. 5. Treatment with pharmacological agents that inhibit calmodulin or disrupt tubulin, two intracellular proteins affected by Cd2+, did not affect CAP area. 6. The effect of Cd2+ was not prevented by pretreatment with (+)-cyanidanol-3, an agent that prevents Cd(2+)-induced lipid peroxidation. 7. Treatment with antimycin A, a inhibitor of mitochondrial respiration, resulted in irreversible CAP reduction with a time course and extent similar to that produced by 200 microM Cd2+. Cd(2+)-induced CAP reduction was prevented by 1 mM cysteine, which prevents Cd(2+)-induced disruption of mitochondrial respiration. 8. The ultrastructure of optic nerves exposed to 200 microM Cd2+ for 100 min was characterized by swollen mitochondria with disrupted cristae and dissolution of microtubules, which were replaced by flocculent debris. Occasional regions of axonal swelling and empty spaces beneath the myelin also were found. Qualitatively similar changes in mitochondria and cytoskeletal elements were found in optic nerves exposed to antimycin A for 100 min. Astrocytes also displayed disrupted mitochondria and had an electron-lucent appearance under both conditions. 9. The neurological injury produced by exposure to Cd2+ is characterized by lesions of CNS white matter. Our results indicate that Cd(2+)-induced white matter injury in vitro results largely from disruption of mitochondrial respiration after Cd2+ influx through routes that include voltage-gated Ca2+ channels.
Animals↗
Voltage-gated calcium channels in CNS white matter: role in anoxic injury.
1. The effect of Ca2+ channel antagonists on the extent of anoxia-induced white matter injury was studied in the rat optic nerve, a white matter tract. Compound action potentials (CAPs) were recorded before and after a standard 60-min anoxic period to assess the extent of anoxic injury. 2. The L-type Ca2+ channel antagonists verapamil (90 microM), diltiazem (50 microM), and nifedepine (2.5 microM) significantly protected the rat optic nerve from anoxic injury. Mean recovery of CAP area was 51.3 +/- 3.0% (mean +/- SE, n = 8, P < 0.01), 65.6 +/- 5.3% (n = 8, P < 0.01), and 54.3 +/- 6.1% (n = 8, P < 0.01), respectively. Mean CAP recovery under control conditions was 35.2 +/- 0.3 (n = 33). 3. Simultaneous block of L-type and N-type Ca2+ channels by coapplication of 50 microM diltiazem and 1 microM SNX-124 [synthetic omega-conotoxin (CgTx) GVIA], resulted in postanoxic CAP recovery of 73.6 +/- 6.0% (n = 12), significantly larger than CAP recovery in diltiazem alone (P < 0.001). Block of CgTx MVIIC-sensitive channels in addition to L-type and N-type channels by coapplication of 50 microM diltiazem + 1 microM SNX-230 + 1 microM SNX-124 failed to produce any additional increase in CAP recovery (71.3 +/- 5.6%, n = 8). Application of 1 microM SNX-124 alone did not significantly protect against anoxic injury (CAP recovery, 36.3 +/- 2.9%, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Endogenous GABA attenuates CNS white matter dysfunction following anoxia.
We studied the effects of GABA on anoxia-induced injury in CNS white matter using optic nerves exposed to 60 min of anoxia. Injury was assessed by recording pre- and postanoxic compound action potentials (CAPs). GABA (1 microM) significantly increased postanoxic CAP recovery when applied 60 min prior to anoxia. This effect was bicuculline (100 microM) insensitive, mimicked by baclofen (1 microM), blocked by GABA-B antagonists, and not mimicked by selective GABA-A agonists. GABA therefore acted at GABA-B receptors. High concentrations of GABA and baclofen did not influence recovery, possibly indicating GABA-B receptor desensitization at high agonist concentrations. Pertussis toxin (PTX) treatment reduced postanoxic CAP recovery in the presence of 1 microM GABA to control levels, indicating the recruitment of a G-protein-linked intracellular pathway. Protein kinase C (PKC) activation with 12-myristate 13-acetate (PMA) mimicked the effects of GABA. Inhibition of PKC with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) or staurosporine reduced postanoxic recovery in the presence of GABA to lower levels than under control conditions, confirming the involvement of PKC in the protective effect of GABA and indicating that this GABA-B receptor/G-protein/PKC protective pathway might be active under control conditions. This was confirmed by the observation that GABA-B receptor blockade, in the absence of exogenous GABA, significantly reduced postanoxia recovery. Thus, activation of the protective mechanism under control conditions is due to endogenous GABA release. Increasing the level of endogenous extracellular GABA by blocking GABA uptake with 1 mM nipecotic acid also protected against anoxia. We propose a model where release of GABA in white matter helps to limit nerve fiber injury during anoxia via recruitment of a G-protein/PKC pathway with subsequent phosphorylation of an unknown target protein.
Animals↗
The relationship between ischaemic conduction failure and conduction velocity in cat myelinated axons.
Unitary action potentials have been recorded from cat spinal root filaments following electrical stimulation of hindlimb peripheral nerves. Potentials were monitored both before the ischaemia which follows the onset of cardiac arrest. The relationship between time to conduction failure (survival time) and initial conduction velocity was determined for 164 nerve fibres. This revealed that during ischaemia, slow myelinated axons tend to fail before fast myelinated axons. The average survival time was 24.6 min (range 11-39 min). The results are discussed in terms of (i) the possible mechanisms underlying ischaemic conduction block, (ii) their clinical implications for the role of ischaemia in peripheral neuropathies, and (iii) using ischaemia as an experimental technique to produce a differential nerve block.
Action Potentials↗
The contribution of ischaemia and deformation to the conduction block generated by compression of the cat sciatic nerve.
The pattern of conduction block induced in cat peripheral nerve by compression has been investigated electrophysiologically by recording unitary action potentials. A degree of compression, associated with chronic compression disorders in man, was applied to the cat sciatic nerve and resulted in a conduction block typified by early failure of the slow conducting myelinated axons. By contrast, a greater degree of compression, a degree more likely to result in acute compression lesions, produced a different pattern of effect, inducing early conduction failure in the fast conducting myelinated axons. These results are related to earlier studies which have shown that nerve deformation during compression has a differential action upon fast myelinated axons, while nerve ischaemia has a differential action upon slow myelinated axons.
Action Potentials↗
Modulation of anoxic injury in CNS white matter by adenosine and interaction between adenosine and GABA.
1. We examined the role of adenosine in the development of anoxic injury in a CNS white matter tract, the rat optic nerve. Application of adenosine protected the rat optic nerve from anoxic injury; 2.5 microM adenosine increased compound action potential (CAP) recovery after a standard 60-min anoxic period from 28.6 +/- 2.5%, mean +/- SE, to 51.0 +/- 3.1% (P < 0001). The protective effect of adenosine was abolished by the adenosine receptor antagonist theophylline (100 microM). 2. The protective effect of adenosine evolved slowly after adenosine application; maximum protection required 60 min of adenosine exposure before the onset of anoxia. The concentration dependence of the protective effect was parabolic, with maximum protection at 2.5 microM. Neither high nor very low adenosine concentrations protected against anoxia. These characteristics are similar to those previously found for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the same preparation. 3. Inhibition of adenosine receptors (100 microM theophylline) reduced the level of recovery from that found under control conditions (24.3 +/- 4.8% compared with 36.2 +/- 2.5%, P < 0.05). The adenosine uptake inhibitor propentofylline, which potentiates release of endogenous adenosine during brain anoxia, significantly increased CAP recovery after anoxia. This effect was abolished by theophylline. It appeared therefore that release of endogenous adenosine limited injury in the optic nerve during anoxia. 4. The protective effect of adenosine was removed by pretreatment with the protein kinase C (PKC) inhibitor staurosporine (10 nM), indicating that activation of PKC was required for protection after exposure to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)
Action Potentials↗
The variation in safety factor with myelinated axon diameter: experiments with low sodium perfusion.
Theoretical considerations suggest that myelinated axons of a small diameter (and therefore with a low conduction velocity) also have a low safety factor. That these axons, many of which are involved in the mediation of pain, may fail first when action potential conduction is inhibited may explain the analgesic properties of local anesthetics. We have tested this hypothesis by perfusing frog myelinated axons with a low concentration of sodium ions and monitoring the reduction in conduction velocity, and the tendency to conduction failure. It was found that axons conducting action potentials at below 10 m/s are affected to the greatest extent by low sodium perfusion, indicating that slow conducting axons do have a low safety factor and confirming the earlier theoretical prediction.
Action Potentials↗
Direct and indirect care in a multidisciplinary collaborative practice model.
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Adult↗