Eric Gilland

Chemokine and Inflammatory Cell Response to Hypoxia-Ischemia in Immature Rats

Hypoxia-ischemia induces an inflammatory response in the immature central nervous system that may be important for development of brain injury. Recent data implicate that chemoattractant cytokines, chemokines, are involved in the recruitment of immune cells. The aim was to study α- and β-chemokines in relation to the temporal activation of inflammatory cells after hypoxia-ischemia in immature rats. Hypoxia-ischemia was induced in 7-day-old rats (left carotid artery occlusion + 7.7% oxygen). The pups were decapitated at different times after the insult. Immunohistochemistry was used for evaluation of the inflammatory cell response and RT-PCR to analyze the cytokine mRNA and chemokine mRNA expression. A distinct interleukin-1β and tumor necrosis factor-α cytokine expression was found 0-24 h after hypoxia-ischemia that was accompanied by induction of α-chemokines (growth related gene and macrophage inflammatory protein-2). In the next phase, the β2-integrin expression was increased (12 h and onward) and neutrophils transiently invaded the vessels and tissue in the infarct region. The mRNA induction for the β-chemokines macrophage inflammatory protein-1α, macrophage inflammatory protein-1β, and RANTES preceded the expression of markers for lymphocytes [cluster of differentiation (CD)4, CD8], microglia/macrophages (MHC I), and natural killer cells in the infarct area. The activation of microglia/macrophages, CD4 lymphocytes, and astroglia persisted up to at least 42 d of postnatal age implicating a chronic component of immunoinflammatory activation. The expression of mRNA for α- and β-chemokines preceded the appearance of immune cells suggesting that these molecules may have a role in the inflammatory response to insults in the immature central nervous system.

Microglia activation after neonatal hypoxic-ischemia

The inflammatory response following hypoxic-ischemia (HI) in the neonate is largely unknown. Presently, the expression of microglial antigens and the beta-amyloid precursor protein (APP) were studied in relation to a dendrosomatic marker of neuronal injury (microtubule associated protein II; MAP II). HI was induced in 7-day-old rats by the combined unilateral carotid ligation and hypoxia. The pups (n = 23) were perfusion fixed 2-3 h, 24 h, 2-4 days and 14 days after HI and compared to sham-operated controls (n = 6). Antibodies were used for detection of the major histocompatibility complex II (OX-6), major histocompatibility complex I (OX-18) and complement receptor type 3 (OX-42), APP (APP 676-695) and MAP II (monoclonal MAP II) antigens. There was a transient APP expression 2-3 h after HI. A slight increase of microglial antigens (OX-18) was seen in the white matter 2 h after HI followed by a marked increase of OX-18, OX-6, OX-42 antigens 24 h-3-4 days in most injured regions with exception of the thalamus where a delayed (14 days) microglial response was seen. The latter event was parallelled by a delayed loss of MAP II. In conclusion, intense microglial expression occurs after neonatal HI either with an acute or delayed time-course depending on brain region.

Hypoxia-ischemia in the neonatal rat brain: histopathology after post-treatment with NMDA and non-NMDA receptor antagonists

In a model of perinatal hypoxic-ischemic brain damage, we examined the neuroprotective efficacy of posttreatment with the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist NBQX. Unilateral brain damage developed in 95% of rat pups subjected to hypoxia-ischemia with a 27.8 +/- 1.2% weight deficit of the damaged hemisphere. MK-801 in doses of 0.3 and 0.5 mg/kg i.p. reduced the brain damage by 61% (p < 0.001) and 43% (p < 0.001), respectively. A higher dose of MK-801 (0.75 mg/kg) did not offer neuroprotection. Treatment with NBQX (40 mg/kg) reduced the hemispheric lesion by 28% (p < 0.05). In conclusion, posttreatment with both NBQX and low doses of MK-801 reduced perinatal brain damage. The NMDA receptor antagonist offered stronger neuroprotection which is in agreement with a proposed NMDA receptor hyperactivity around postnatal day 7 in rats.

Impairment of mitochondrial respiration after cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury.

Mitochondrial damage may play a key role in the development of necrotic and apoptotic hypoxic-ischemic (HI) brain damage. It has previously been shown that mitochondrial respiration is depressed in the cerebral cortex after HI in neonatal animals. The aim of the present study was to further characterize the time course of the mitochondrial impairment during reperfusion and the correlation between the respiratory control ratio and brain injury and activation of caspase-3. Rat pups were subjected to unilateral carotid artery ligation and exposed to hypoxia (7.7% oxygen). Mitochondrial respiration was measured 0-72 h after HI in a mitochondrial fraction isolated from cerebral cortex. Microtubule associated protein-2 (MAP2) and caspase-3 were analyzed with immunoblotting in cerebral cortex homogenates. In addition, the time course of caspase-3 activation was measured as DEVD cleavage. The mitochondrial respiratory control ratio in cerebral cortex decreased immediately after HI followed by a partial recovery at 3-8 h. Thereafter, a secondary drop occurred with a minimum reached at 24 h of reperfusion. The secondary loss of respiratory function was accompanied by depletion of MAP2, cleavage of caspase-3 and an increased caspase-3 -like activity at 3-24 h after the insult. In conclusion, the primary phase of mitochondrial dysfunction was paralleled by a moderate decrease of MAP2 and a limited activation of caspase-3. The secondary mitochondrial impairment was associated with neuronal injury and pronounced activation of caspase-3.

Mitochondrial Function and Energy Metabolism after Hypoxia—Ischemia in the Immature Rat Brain: Involvement of NMDA-Receptors

Treatment after hypoxia—ischemia (HI) in immature rats with the N-methyl-d-aspartate receptor (NMDAR) antagonist dizocilpine maleate (MK-801) reduces areas with high glucose utilization and reduces brain damage. The object was to study the metabolic effects of MK-801 treatment after HI. Seven-day-old rats were randomized to the following groups: non-HI, HI, or HI plus MK-801 (0.5 mg/kg immediately after HI). In the parietal cortex, the mitochondrial respiration was measured in homogenates 1 to 4 hours, and the energy metabolites at 3 and 8 hours after HI. The energy use was calculated from changes in energy metabolites after decapitation at 3 hours after HI. State 3 respiration was reduced by 46%, 32%, and 25% after HI compared with non-HI with pyruvate plus malate, glutamate plus malate, or glutamate plus succinate as substrates, respectively. Uncoupler-stimulated but not state 4 respiration was similarly reduced. The MK-801 augmented pyruvate plus malate—supported state 3 respiration after HI by 42%. The energy utilization was not affected by HI but was reduced by MK-801 treatment in the ipsilateral cortex from 4.6 ± 2.3 to 2.6 ± 1.8 μmol ~P/min/g. The levels of ATP and phosphocreatine did not differ between the HI and HI plus MK-801 groups at 3 hours, but were lower in the HI than in the HI plus MK-801 group at 8 hours after HI. In conclusion, treatment with MK-801 reduced energy utilization and improved mitochondrial function and energy status after HI, suggesting a linkage between NMDAR activation and impaired energy metabolism during reperfusion.

Temporal Changes of Regional Glucose Use, Blood Flow, and Microtubule-Associated Protein 2 Immunostaining After Hypoxia-Ischemia in the Immature Rat Brain

In a situation with normal CBF and without increased energy utilization, increased glucose utilization (CMRglc) can be a sign of impaired mitochondrial metabolism, which may be an early step in the injury cascade during reperfusion after hypoxia–ischemia (HI). Seven-day-old rats underwent unilateral carotid artery ligation and 70 minutes of HI. At 3, 6, 12, 24, and 48 or 72 hours after the insult, the CMRglc was measured by the 2-deoxyglucose method, and CBF by the iodoantipyrine method. These were compared with hematoxylin-eosin staining and microtubule-associated protein 2 (MAP 2) immunostaining in adjacent sections. In the ipsilateral hemisphere, there appeared regions with increased CMRglc compared with the contralateral hemisphere 3 to 12 hours after HI that also showed partial loss of MAP 2 immunostaining and early ischemic changes. These areas receded, leaving central glucose hypoutilizing areas with complete loss of MAP 2 immunostaining and histologic infarction, surrounded by only a rim of tissue with increased CMRglc. At 24 and 72 hours after the insult, no regions with increased CMRglc remained. Despite loss of MAP 2 immunostaining and histologic signs of infarction at 24 hours, cortical CBF was not reduced until 48 hours after HI, whereas the CBF in the caudate-putamen already was decreased compared with the contralateral side at 3 hours after HI. In conclusion, early reperfusion is characterized by glucose hyperutilizing areas in the cerebral cortex, followed by a secondary phase with low CMRglc and infarction.

NMDA Receptor-Dependent Increase of Cerebral Glucose Utilization After Hypoxia-Ischemia in the Immature Rat

Post-treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces hypoxic–ischemic brain injury in immature animals. To elucidate possible mechanisms, cerebral glucose utilization (CMRglc) and cerebral blood flow (CBF) were measured 1–5 h after hypoxia–ischemia and administration of MK-801 in 7-day-old rats. After 100 min of unilateral hypoxia–ischemia, half of the pups were injected with MK-801. CMRglc was assessed by the [14C]deoxyglucose (2-DG) method. The brains were analyzed either by autoradiography or for energy metabolites and chromatographic separation of 2-DG-6-phosphate and 2-DG. CBF was measured by the autoradiographic [14C]iodoantipyrine method. Mean CMRglc in the cerebral cortex was increased ipsilaterally after hypoxia–ischemia to 15 ± 3.3 μmol 100 g−1 min−1 (p < 0.01) and areas with CMRglc >20 μmol 100 g−1 min−1 amounted to 8.0 ± 7.7 mm2 in the ipsilateral hemisphere compared with 1.2 ± 1.6 mm2 contralateral (p < 0.001). Treatment with MK-801 decreased CMRglc bilaterally (p < 0.05) and reduced ipsilateral areas with increased CMRglc by 64% (p < 0.01). CBF was unaltered after hypoxia–ischemia and by MK-801 treatment. In conclusion, regional glucose hyper-utilization in the parietal cortex after hypoxia–ischemia was attenuated by MK-801; this may have relevance to the neuroprotective effect of NMDA-receptor antagonists in this model.

Cerebral hypoxia-ischemia in immature rats: involvement of mitochondrial permeability transition?

The aim of this study was to evaluate the involvement of mitochondrial membrane permeability transition (MPT) after hypoxia-ischemia (HI) in 7-day-old rats. [14C]2-deoxyglucose (DOG) was administered to controls, and at various time points after HI. MPT in the cerebral cortex was measured as entrapment of DOG-6-P in mitochondria. Another group of rats was treated with the MPT inhibitor cyclosporin A (CsA; 10–50 mg/kg i.p.) or vehicle before and after HI, and the effect on brain injury and mitochondrial respiration was evaluated. A significant increase in DOG-6-P entrapment in mitochondria indicated that MPT occurred in two phases: a primary MPT after 0–1.5 h and a secondary MPT after 6.5–8 h of reperfusion. However, CsA did not affect brain injury or mitochondrial respiration. The data suggest that MPT occurred after HI but does not provide evidence for its involvement in the development of injury.

Hypoxic-ischemic injury in the neonatal rat brain: effects of pre- and post-treatment with the glutamate release inhibitor BW1003C87.

In a model of perinatal hypoxia-ischemia (HI) we examined the neuroprotective efficacy of pre- and post-treatment with the glutamate release inhibitor BW1003C87 [5-(2,3,5-trichlorophenyl)-2,4-diamino-pyrimidine). Ipsilateral brain damage developed in 99% of rat pups subjected to HI (unilateral common carotid artery ligation and 100 min of 7.7% oxygen exposure) with a 26 +/- 16% (mean +/- S.D.) weight deficit of the damaged hemisphere 2 weeks after the insult. Pre-treatment with BW1003C87 (10 mg/kg intraperitoneally) reduced the brain damage by 46% (P < 0.05). A higher dose (20 mg/kg) of pre-treatment was not tolerated. Administration of BW1003C87 did not affect the rectal temperature of the rats. Post-treatment with BW1003C87 (10-30 mg/kg) offered no neuroprotection in this model. In conclusion, there was a neuroprotective effect from pre- but not post-treatment with BW1003C87 in this model, supporting the concept that intra-ischemic excitatory amino acid release is important for development of brain damage. The lack of post-treatment effect indicates that BW1003C87 did not attenuate deleterious EAA cycling during reflow in the neonatal brain.

Is MK-801 neuroprotection mediated by systemic hypothermia in the immature rat?

HYPOTHERMIA after hypoxia–ischaemia (HI) confounds the interpretation of the effects of neuroprotective drug intervention. The effect of 0.5 mg/kg of dizocilpine (MK-801) administered after HI on rectal temperature at 2–36 h and on brain damage 2 weeks after the insult was evaluated in the immature rat. In pups kept at an ambient temperature of 21°C, MK-801 lowered the temperature by 1.1°C and reduced the brain damage by 45%. In pups held at an ambient temperature of 33°C, MK-801 treatment afforded a 34% reduction of brain damage without lowering the rectal temperature. In conclusion, the neuroprotection offered by MK-801 does not depend on systemic hypothermia in this model.