Elsa Bona

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.

Sensorimotor Function and Neuropathology Five to Six Weeks after Hypoxia-Ischemia in Seven-Day-Old Rats

Various therapeutic interventions after hypoxia-ischemia (HI) have been shown to reduce brain injury in the short-term perspective, but it remains uncertain whether such findings are accompanied by long-term functional and structural improvements. HI was induced in 7-d-old rats as follows. The left carotid artery was ligated, and the rat was exposed to 100 min of hypoxia (7.70% oxygen in nitrogen). At postnatal d 42 the rats were assessed using four sensorimotor tests. The results were correlated with the extent of brain damage expressed as volume of deficit of the left hemisphere as percent of the right hemisphere. In the grip-traction test, the time to falling was 2.2 times shorter in the HI animals compared with controls (p < 0.01). Asymmetries of limb-placing and foot-faults (p < 0.001) were detected in HI animals, and the motor function was abnormal in the postural reflex test (p < 0.001). We found a moderate correspondence between functional and neuropathologic outcome (r = 0.842, p < 0.001). A set of four easily performed sensorimotor tests is presented for the long-term evaluation of neurologic function in the 7-d-old rat model of HI.

Neonatal cerebral hypoxia-ischemia: The effect of adenosine receptor antagonists

The effects of nonselective (theophylline), A1-(DPCPX) or A2A-selective (SCH 58261) adenosine receptor antagonists administered before or after neonatal hypoxia-ischemia (HI) were studied on the extent of brain injury in 7-day-old rats evaluated after 14 days. A possible effect of theophylline (20 mg/kg) on expression of immediate early genes was studied with in situ hybridization. Theophylline (20, 30 or 60 mg/kg) given prior to HI reduced brain damage by 48% (P < 0.001), 36% (P < 0.01) and 34% (P < 0.05), respectively, compared to control rats. This effect was not explained by changes in temperature, cerebral blood flow, blood gas/acid base status or blood glucose during the insult. Theophylline enhanced the upregulation of c-fos and NFGI-A during reperfusion but did not prevent the decrease in adenosine A1 receptor mRNA. Posttreatment with SCH 58261 (0.2 or 2 mg/kg) reduced brain damage by 19% (P < 0.05) and 14% (NS), respectively, compared to control rats which was unrelated to the core temperature. DPCPX (2 or 10 mg/kg) had no effect on the development of brain injury. In conclusion, nonselective and A2A adenosine receptor antagonists reduced brain injury in a model of HI in immature animals.

The effect of long term caffeine treatment on hypoxic-ischemic brain damage in the neonate.

ABSTRACT: There is considerable concern over the widespread use of caffeine during and after pregnancy. We have therefore examined the effect of perinatal caffeine use on the vulnerability of the immature brain to hypoxic ischemia (HI). Rat pups were exposed to caffeine during the first 7 d after birth by addition of a low or a high dose (0.3 or 0.8 g/L) of caffeine to the drinking water of their dams. At 7 d the pups were exposed to unilateral carotid occlusion + exposure to 7.70% oxygen for 100 min. The extent of HI brain damage was evaluated 2 wk after the insult. The effects of caffeine on A1 and A2a receptors, A1 mRNA and A2a mRNA, were examined by receptor autoradiography and in situ hybridization. Caffeine, theobromine, theophylline, and paraxanthine were analyzed in plasma of separate animals. Exposure to caffeine reduced HI brain damage from 40.3 ± 3.2% in controls to 29.8 ± 4.0% (p < 0.05) in low dose and 33.7 ± 3.9% (NS) in the high dose group. The A1 receptor density measured as [3H]-l,3-dipropyl-8-cyclopentyl xanthine ([3H]-DPCPX) binding was not significantly affected after low dose caffeine but increased in the brain of rat pups in the high dose group. The A2a receptor density measured as [3H]-2[p-(2-carbonylethyl)-phenethylamino]-5′-N-ethylcarboxamidoadenosine ([3H]-CGS 21680) binding and the expression of A1 mRNA and A2a mRNA were not altered by caffeine treatment. In conclusion, low dose caffeine exposure (plasma levels corresponding to umbilical cord plasma in newborns of coffee-consuming mothers) reduced HI brain damage by 30% in 7-d-old rats. This ameliorating effect could not be accounted for by up-regulation of adenosine receptors.

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.

Degradation of fodrin and MAP 2 after neonatal cerebral hypoxic-ischemia

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min) and allowed to recover for 3 h, 24 h, 2 days or 14 days. Consecutive tissue sections were stained with antibodies against alpha-fodrin, the 150 kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule associated protein 2 (MAP 2, marker of dendrosomatic neuronal injury). Cortical tissue pieces were subjected to Western blotting using the antibody against the FBDP. Areas with brain injury displayed a distinct loss of MAP 2 which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. A reciprocal staining pattern could be seen in the cerebral cortex, i.e. loss of MAP 2 and accumulation of FBDP, most pronounced 14 days after the insult. Fodrin and MAP 2 are known calpain substrates, and degradation of these proteins preceded neuronal degeneration, indicating that these proteases may be involved in the early events triggering the cascades leading to neuronal death.

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.

Changes in adenosine receptors in the neonatal rat brain following hypoxic ischemia

We used quantitative in situ hybridization and receptor autoradiography to study changes in adenosine receptors following hypoxia-ischemia (H-I) in the neonatal rat brain. Seven-day-old rat pups were subjected to a unilateral ligation of the common carotid artery followed by a 2 h 15 min hypoxic period (7.7% O2 in N2). Adenosine A1 receptor mRNA in cortex and several parts of hippocampus, and A2a mRNA was decreased in the ligated hemisphere 0 h, 1 h and 2 h following hypoxia. The binding of the A1 receptor selective antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in the presence or in the absence of GTP decreased immediately after the hypoxic period in both hemispheres and returned thereafter gradually towards control. These results show that there are rapid changes in A1 receptor number on both sides of the brain, and of adenosine A1 and A2a receptor mRNA in the hemisphere that would later develop infarction. Decreases in adenosine receptors may worsen H-I brain damage and have consequences for the use of adenosine directed therapy.

The Calpain Proteolytic System in Neonatal Hypoxic‐Ischemiaa

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.