Microglial depletion abolishes ischemic preconditioning in white matter.

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.

A method for reducing animal use whilst maintaining statistical power in electrophysiological recordings from rodent nerves.

The stimulus evoked compound action potential, recorded from ex vivo nerve trunks such as the rodent optic and sciatic nerve, is a popular model system used to study aspects of nervous system metabolism. This includes (1) the role of glycogen in supporting axon conduction, (2) the injury mechanisms resulting from metabolic insults, and (3) to test putative benefits of clinically relevant neuroprotective strategies. We demonstrate the benefit of simultaneously recording from pairs of nerves in the same superfusion chamber compared with conventional recordings from single nerves. Experiments carried out on mouse optic and sciatic nerves demonstrate that our new recording configuration decreased the relative standard deviation from samples when compared with recordings from an equivalent number of individually recorded nerves. The new method reduces the number of animals required to produce equivalent Power compared with the existing method, where single nerves are used. Adopting this method leads to increased experimental efficiency and productivity. We demonstrate that reduced animal use and increased Power can be achieved by recording from pairs of rodent nerve trunks simultaneously.

Ischemic Preconditioning in White Matter: Magnitude and Mechanism.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon whereby brief ischemic exposure confers tolerance to a subsequent ischemic challenge. IPC has not been studied selectively in CNS white matter (WM), although stroke frequently involves WM. We determined whether IPC is present in WM and, if so, its mechanism. We delivered a brief in vivo preconditioning ischemic insult (unilateral common carotid artery ligation) to 12- to 14-week-old mice and determined WM ischemic vulnerability [oxygen-glucose deprivation (OGD)] 72 h later, using acutely isolated optic nerves (CNS WM tracts) from the preconditioned (ipsilateral) and control (contralateral) hemispheres. Functional and structural recovery was assessed by quantitative measurement of compound action potentials (CAPs) and immunofluorescent microscopy. Preconditioned mouse optic nerves (MONs) showed better functional recovery after OGD than the non-preconditioned MONs (31 ± 3 vs 17 ± 3% normalized CAP area, p < 0.01). Preconditioned MONs also showed improved axon integrity and reduced oligodendrocyte injury compared with non-preconditioned MONs. Toll-like receptor-4 (TLR4) and type 1 interferon receptor (IFNAR1), key receptors in innate immune response, are implicated in gray matter preconditioning. Strikingly, IPC-mediated WM protection was abolished in both TLR4(-/-) and IFNAR1(-/-) mice. In addition, IPC-mediated protection in WM was also abolished in IFNAR1(fl/fl) LysM(cre), but not in IFNAR1(fl/fl) control, mice. These findings demonstrated for the first time that IPC was robust in WM, the phenomenon being intrinsic to WM itself. Furthermore, WM IPC was dependent on innate immune cell signaling pathways. Finally, these data demonstrated that microglial-specific expression of IFNAR1 plays an indispensable role in WM IPC. SIGNIFICANCE STATEMENT: Ischemic preconditioning (IPC) has been studied predominantly in gray matter, but stroke in humans frequently involves white matter (WM) as well. Here we describe a novel, combined in vivo/ex vivo mouse model to determine whether IPC occurs in WM. It does. Using genetically altered mice, we identified two innate immune cell receptors, Toll-like receptor 4 and type 1 interferon receptor (IFNAR1), that are required for IPC-mediated protection in WM. Furthermore, using microglia-targeted IFNAR1 knockdown, we demonstrate that interferon signaling specifically in microglia is essential for this protection. The discovery of IPC as an intrinsic capability of WM is novel and important. This is also the first in vivo demonstration that cell-type-specific expression of an individual gene plays an indispensable role in IPC-mediated protection.

Novel hypoglycemic injury mechanism: N-methyl-D-aspartate receptor-mediated white matter damage.

OBJECTIVE: Hypoglycemia is a common adverse event and can injure central nervous system (CNS) white matter (WM). We determined whether glutamate receptors were involved in hypoglycemic WM injury. METHODS: Mouse optic nerves (MON), CNS WM tracts, were maintained at 37°C with oxygenated artificial cerebrospinal fluid (ACSF) containing 10mM glucose. Aglycemia was produced by switching to 0 glucose ACSF. Supramaximal compound action potentials (CAPs) were elicited using suction electrodes, and axon function was quantified as the area under the CAP. Amino acid release was measured using high-performance liquid chromatography. Extracellular lactate concentration ([lactate(-)]o) was measured using an enzyme electrode. RESULTS: About 50% of MON axons were injured after 60 minutes of aglycemia (90% after 90 minutes); injury extent was not affected by animal age. Blockade of N-methyl-D-aspartate (NMDA)-type glutamate receptors improved recovery after 90 minutes of aglycemia by 250%. Aglycemic injury was increased by reducing [Mg(2+)]o or increasing [glycine]o , and decreased by lowering pHo , expected results for NMDA receptor-mediated injury. pHo increased during aglycemia due to a drop in [lactate(-)]o. Aglycemic injury was dramatically reduced in the absence of [Ca(2+)]o. Extracellular aspartate, a selective NMDA receptor agonist, increased during aglycemia ([glutamate]o fell). INTERPRETATION: Aglycemia injured WM by a unique excitotoxic mechanism involving NMDA receptors (located primarily on oligodendrocytes). During WM aglycemia, the selective NMDA agonist aspartate is released, probably from astrocytes. Injury is mediated by Ca(2+) influx through aspartate-activated NMDA receptors made permeable by an accompanying alkaline shift in pHo caused by a fall in [lactate(-)]o. These insights have important clinical implications.

Anaerobic function of CNS white matter declines with age.

The mammalian central nervous system (CNS) is generally believed to be completely dependent on the presence of oxygen (O(2)) to maintain energy levels necessary for excitability. However, previous studies on CNS white matter (WM) have shown that a large subset of CNS-myelinated axons of mice aged 4 to 6 weeks remains excitable in the absence of O(2). We investigated whether this surprising WM tolerance to anoxia varied with age. Acutely isolated mouse optic nerve (MON), a purely myelinated WM tract, was studied electrophysiologically. Excitability in the MONs from 1-month-, 4-month-, and 8-month-old mice was assessed quantitatively as the area under the supramaximal compound action potential (CAP). Anoxia-resistant WM function declined with age. After 60 minutes of anoxia, ∼23% of the CAP remained in 1-month-old mice, 8% in 4-month-old mice, and ∼0 in the 8-month-old group. Our results indicated that although some CNS axons function anaerobically in young adult animals, they lose this ability in later adulthood. This finding may help explain the clinical impression that favorable outcome after stroke and other brain injuries declines with age.

White matter vulnerability to ischemic injury increases with age because of enhanced excitotoxicity.

Stroke incidence increases with age and this has been attributed to vascular factors. We show here that CNS white matter (WM) is intrinsically more vulnerable to ischemic injury in older animals and that the mechanisms of WM injury change as a function of age. The mouse optic nerve was used to study WM function. WM function in older animals (12 months) was not protected from ischemic injury by removal of extracellular Ca2+ or by blockade of reverse Na+/Ca2+ exchange, as is the case with young adults. Ischemic WM injury in older mice is predominately mediated by glutamate release and activation of AMPA/kainate-type glutamate receptors. Glutamate release, attributable to reverse glutamate transport, occurs earlier and is more robust in older mice that show greater expression of the glutamate transporter. The observation that WM vulnerability to ischemic injury is age dependent has possible implications for the pathogenesis of other age-related CNS conditions.

Elongation and secretion of tissue inhibitor of metalloproteinases 1 by human microvascular endothelial cells cultured in collagen gels is stimulated by mitomycin c.

During angiogenesis, interactions between endothelial cells (ECs) and the surrounding extracellular matrix are influenced by matrix metalloproteinases (MMPs) and their cognate inhibitors, the TIMPs. The authors discovered that the secretion of TIMP-1 by human microvascular ECs (hmECs) cultured within gels of native, fibrillar collagen was increased robustly by mitomycin C (MMC), an inhibitor of cell proliferation. In contrast, hmECs cultured on plastic coated with gelatin or with native fibrillar collagen exhibited nil (on gelatin) or very modest (on native collagen) increases in TIMP-1 upon exposure to MMC. Notably, none of the cultures altered the secretion of TIMP-2, or MMP-1 and -2, in response to MMC. hmECs cultured within collagen gels elongated significantly after exposure to MMC, a response the authors concluded was mediated by TIMP-1, because elongation could be inhibited completely with a function-blocking antibody to TIMP-1. Moreover, substitution of purified human TIMP-1 for MMC induced a similar elongation by hmECs. hmECs cultured within collagen gels did not proliferate under the conditions used in this study; therefore, inhibited proliferation was not a factor in the altered cell shape and TIMP-1 secretion elicited by MMC. These results illustrate that antiproliferative compounds should be used with caution in studies of MMP regulation by ECs.

MT1-MMP, but not secreted MMPs, influences the migration of human microvascular endothelial cells in 3-dimensional collagen gels.

Matrix metalloproteinases (MMPs) and their specific inhibitors the TIMPs play significant roles in angiogenesis. We investigated how the expression of specific MMPs and TIMPs by human microvascular endothelial cells (hmECs) was modulated by culture of the cells in 3-dimensional (3D) type I collagen gels versus 2-dimensional (2D) collagen-coated surfaces. By reverse-transcription polymerase chain reaction (RT-PCR), levels of mRNA for MMPs-1, -2, and -13, MT1-MMP, and TIMPs-1 and -2 were similar in 2D versus 3D cultures. By Western blot assay, TIMP-1 and proMMP-1 were present and were expressed similarly in media from 2D versus 3D cultures, whereas active MMPs-1, -9, and -13 were not detected. Active MMP-13 was present in cell lysates (CL) and was increased in lysates from 3D cultures relative to 2D cultures. Relative to 2D cultures, CL and media from 3D cultures exhibited a decrease in expression of TIMP-2 and an increased conversion of proMMP-2 and proMT1-MMP to active or processed forms. The MMP inhibitor GM6001 interfered with the migration of hmECs in 3D cultures, but not in 2D cultures. Addition of active MMP-1 or blocking antibodies to TIMP-1 did not affect the migration of hmECs in 3D collagen. Migration in 3D collagen was decreased by TIMP-2 (an inhibitor of MT1-MMP), but not by TIMP-1 (a poor inhibitor of MT1-MMP, but an efficient inhibitor of MMP-2). Collectively, our data indicate that MT1-MMP contributes significantly to the movement of hmECs through 3D collagen, in contrast to secretory-type MMPs-1, -2, -9, and -13, which are not critical for this movement.