Cora H. Nijboer

Extent of bilateral neuronal network reorganization and functional recovery in relation to stroke severity.

Remodeling of neuronal structures and networks is believed to significantly contribute to (partial) restoration of functions after stroke. However, it has been unclear to what extent the brain reorganizes and how this correlates with functional recovery in relation to stroke severity. We applied serial resting-state functional MRI and diffusion tensor imaging together with behavioral testing to relate longitudinal modifications in functional and structural connectivity of the sensorimotor neuronal network to changes in sensorimotor function after unilateral stroke in rats. We found that gradual improvement of functions is associated with wide-ranging changes in functional and structural connectivity within bilateral neuronal networks, particularly after large stroke. Both after medium and large stroke, brain reorganization eventually leads to (partial) normalization of neuronal signal synchronization within the affected sensorimotor cortical network (intraregional signal coherence), as well as between the affected and unaffected sensorimotor cortices (interhemispheric functional connectivity). Furthermore, the bilateral network configuration shifts from subacutely increased “small-worldness,” possibly reflective of initial excessive neuronal clustering and wiring, toward a baseline small-world topology, optimal for global information transfer and local processing, at chronic stages. Cortical network remodeling was accompanied by recovery of initially disrupted structural integrity in corticospinal tract regions, which correlated positively with retrieval of sensorimotor functions. Our study demonstrates that the degree of functional recovery after stroke is associated with the extent of preservation or restoration of ipsilesional corticospinal tracts in combination with reinstatement of interhemispheric neuronal signal synchronization and normalization of small-world cortical network organization.

Strong Neuroprotection by Inhibition of NF-κB After Neonatal Hypoxia-Ischemia Involves Apoptotic Mechanisms but Is Independent of Cytokines

Background and Purpose— Interactions between excitotoxic, inflammatory, and apoptotic pathways determine outcome in hypoxic-ischemic brain damage. The transcription factor NF-&kgr;B has been suggested to enhance brain damage via stimulation of cytokine production. There is also evidence that NF-&kgr;B activity is required for neuronal survival. We used the NF-&kgr;B inhibitor NBD, coupled to TAT to facilitate cerebral uptake, to determine the neuroprotective capacity of NF-&kgr;B inhibition in neonatal hypoxia-ischemia (HI) and to identify its contribution to cerebral inflammation and damage. Methods— Brain damage was induced in neonatal rats by unilateral carotid artery occlusion and hypoxia and analyzed immunohistochemically; NF-&kgr;B activity was analyzed by EMSA. We analyzed cytokine mRNA levels and activation of apoptotic pathways by Western blotting. In vitro effects of TAT-NBD were determined in a neuronal cell line. Results— Inhibition of cerebral NF-&kgr;B activity by TAT-NBD had a significant neuroprotective effect; brain damage was reduced by more than 80% with a therapeutic window of at least 6 hours. In contrast to earlier suggestions, the protective effect of TAT-NBD did not involve suppression of early cytokine upregulation after HI. Moreover, NF-&kgr;B inhibition prevented HI-induced upregulation and nuclear as well as mitochondrial accumulation of p53, prevented mitochondrial cytochrome-c release and activation of caspase-3. Finally, TAT-NBD could directly increase neuronal survival because TAT-NBD was sufficient to inhibit death in a neuronal cell line. A nonactive mutant peptide did not have any effect. Conclusions— Inhibition of NF-&kgr;B has strong neuroprotective effects that involve downregulation of apoptotic molecules but are independent of inhibition of cytokine production.

Gender-Specific Neuroprotection by 2-Iminobiotin after Hypoxia—Ischemia in the Neonatal Rat via a Nitric Oxide Independent Pathway

We have shown earlier that 2-iminobiotin (2-IB) reduces hypoxia-ischemia (HI)-induced brain damage in neonatal rats, and presumed that inhibition of nitric oxide synthases (NOS) was the underlying mechanism. We now investigated the effect of 2-IB treatment in P7 rat pups to determine the role of gender and the neuroprotective mechanism. Pups were subjected to HI (occlusion of right carotid artery and 120 mins FiO2 0.08) and received subcutaneous (s.c.) 10 mg/kg 2-IB at 0, 12 and 24 h after hypoxia. After 6 weeks, neuronal damage was assessed histologically. We determined cerebral nitrite and nitrate (NOx) and nitrotyrosine, heat-shock protein 70, cytosolic cytochrome c, cleaved caspase 3, nuclear translocation of apoptosis-inducing factor (AIF) and the effect of 2-IB on NOS activity in cultured cells. 2-Iminobiotin treatment reduced long-term brain damage in female but not male rats. Unexpectedly, 2-IB treatment did not reduce cerebral NOx or nitrotyrosine levels, and did not inhibit NOS activity in vitro. The gender-dependent neuroprotective effect of 2-IB was reflected in inhibition of the HI-induced increase in cytosolic cytochrome c and cleaved caspase 3 in females only. Hypoxia–ischemia-induced activation of AIF was observed in males only and was not affected by 2-IB. Post-HI treatment with 2-IB provides gender-specific long- and short-term neuroprotection in female P7 rats via inhibition of the cytochrome c-caspase 3 neuronal death pathway. 2-Iminobiotin did not alter cerebral NOx nor inhibited NOS in intact cells. Therefore, we conclude that it is highly unlikely that the neuroprotective effect of 2-IB involves NOS inhibition.

Intranasal Mesenchymal Stem Cell Treatment for Neonatal Brain Damage: Long-Term Cognitive and Sensorimotor Improvement

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5×106 MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.

A Dual Role of the NF-κB Pathway in Neonatal Hypoxic-Ischemic Brain Damage

Background and Purpose— NF-&kgr;B is a transcription factor that regulates inflammatory and apoptotic pathways. We described previously that intraperitoneal administration of the NF-&kgr;B inhibitor TAT-NBD at 0 and 3 hours after neonatal hypoxia-ischemia (HI) markedly reduced brain damage. We hypothesize that timing and duration of NF-&kgr;B inhibition will be a major factor in determining outcome. Methods— HI was induced in P7 rats by unilateral carotid artery occlusion and hypoxia. In vivo TAT-NBD effects were determined on cerebral damage, NF-&kgr;B activity, cytokine expression, and pro- and antiapoptotic molecules. In vitro effects of TAT-NBD were determined using primary neurons and cell lines. Results— HI induced 2 peaks of cerebral NF-&kgr;B activity at 3 to 6 and 24 hours after HI. Neuroprotective 0/3-hour TAT-NBD treatment only inhibited early NF-&kgr;B activity. However, inhibition of both early and late NF-&kgr;B-activity by 0/6/12-hour TAT-NBD or only late NF-&kgr;B activity by 18/21-hour TAT-NBD aggravated damage. 0/6/12-hour TAT-NBD did not prevent HI-induced upregulation of cytokines at 24 hours after HI. Protective 0/3-hour TAT-NBD treatment prevented nuclear accumulation of p53 at 24 hours after HI. Nuclear p53 was not reduced after 0/6/12-hour TAT-NBD. Prolonged TAT-NBD increased the proapoptotic factor PUMA and reduced the antiapoptotic factors Bcl-2 and Bcl-xL. Also in neuronal cultures prolonged TAT-NBD exposure overruled protective short-term TAT-NBD treatment. Conclusions— Early NF-&kgr;B activation contributes to neonatal HI brain damage. Late NF-&kgr;B provides endogenous neuroprotection and upregulates antiapoptotic molecules. Inhibition of early NF-&kgr;B activity is neuroprotective only when late NF-&kgr;B activity is maintained. Moreover, cerebral cytokine production can occur independently of NF-&kgr;B.

Targeting the p53 pathway to protect the neonatal ischemic brain

To investigate whether inhibition of mitochondrial p53 association using pifithrin‐μ (PFT‐μ) represents a potential novel neuroprotective strategy to combat perinatal hypoxic‐ischemic (HI) brain damage.

GRK2: A Novel Cell-Specific Regulator of Severity and Duration of Inflammatory Pain

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2+/− mice with an ∼50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre–Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Nav1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2+/− mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2+/− mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor α (TNF-α) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-α activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2+/− mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.

Intranasally administered mesenchymal stem cells promote a regenerative niche for repair of neonatal ischemic brain injury

Previous work from our group has shown that intranasal MSC-treatment decreases lesion volume and improves motor and cognitive behavior after hypoxic-ischemic (HI) brain damage in neonatal mice. Our aim was to determine the kinetics of MSC migration after intranasal administration, and the early effects of MSCs on neurogenic processes and gliosis at the lesion site. HI brain injury was induced in 9-day-old mice and MSCs were administered intranasally at 10days post-HI. The kinetics of MSC migration were investigated by immunofluorescence and MRI analysis. BDNF and NGF gene expression was determined by qPCR analysis following MSC co-culture with HI brain extract. Nestin, Doublecortin, NeuN, GFAP, Iba-1 and M1/M2 phenotypic expression was assessed over time. MRI and immunohistochemistry analyses showed that MSCs reach the lesion site already within 2h after intranasal administration. At 12h after administration the number of MSCs at the lesion site peaks and decreases significantly at 72h. The number of DCX(+) cells increased 1 to 3days after MSC administration in the SVZ. At the lesion, GFAP(+)/nestin(+) and DCX(+) expression increased 3 to 5days after MSC-treatment. The number of NeuN(+) cells increased within 5days, leading to a dramatic regeneration of the somatosensory cortex and hippocampus at 18days after intranasal MSC administration. Interestingly, MSCs expressed significantly more BDNF gene when exposed to HI brain extract in vitro. Furthermore, MSC-treatment resulted in the resolution of the glial scar surrounding the lesion, represented by a decrease in reactive astrocytes and microglia and polarization of microglia towards the M2 phenotype. In view of the current lack of therapeutic strategies, we propose that intranasal MSC administration is a powerful therapeutic option through its functional repair of the lesion represented by regeneration of the cortical and hippocampal structure and decrease of gliosis.

Inhibition of the JNK/AP-1 pathway reduces neuronal death and improves behavioral outcome after neonatal hypoxic-ischemic brain injury

Perinatal hypoxic-ischemic (HI) brain damage continues to be a major clinical problem. We investigated the contribution of the MAP kinase c-Jun N-terminal kinase (JNK), to neonatal HI brain damage. JNK regulates several transcriptional (via AP-1 activation) and non-transcriptional processes involved in brain damage such as inflammation and cell death/survival. P7 rats were subjected to HI by unilateral carotid artery occlusion and hypoxia. HI-induced activation of cerebral AP-1 peaked at 3-6h post-HI. Intraperitoneal administration of the JNK-inhibitor TAT-JBD immediately after HI prevented AP-1 activation. TAT-JBD treatment within 3h after HI reduced early neuronal damage by approximately 30%. JNK/AP-1 inhibition did not reduce HI-induced cytokine/chemokine expression. Analysis of indicators of apoptotic cell death revealed that TAT-JBD markedly reduced the HI-induced increase in active caspase 3. However, the upstream mediators of apoptosis: active caspase 8, cleaved Bid, mitochondrial cytochrome c release and caspase 9 cleavage were not reduced after TAT-JBD. TAT-JBD inhibited the HI-induced increase in Smac/DIABLO, an inhibitor of IAPs that prevent activation of caspase 3. TAT-JBD treatment also reduced cleavage of alpha-fodrin, indicating that calpain-mediated brain damage was reduced. Neuroprotection by TAT-JBD treatment was long-lasting as gray- and white matter damage was diminished by approximately 50% at 14 weeks post-HI concomitantly with marked improvement of sensorimotor behavior and cognitive functioning. In conclusion, JNK inhibition by TAT-JBD treatment reduced neonatal HI brain damage with a therapeutic window of 3h and long-lasting anatomical and behavioral improvements. We propose that inhibition of mitochondrial Smac/DIABLO release and calpain activation contribute to neuroprotection by TAT-JBD.

Bilateral molecular changes in a neonatal rat model of unilateral hypoxic-ischemic brain damage.

Perinatal hypoxia ischemia (HI) is a frequent cause of neonatal brain injury. This study aimed at describing molecular changes during the first 48 h after exposure of the neonatal rat brain to HI. Twelve-day-old rats were subjected to unilateral carotid artery occlusion and 90 min of 8% O2, leading to neuronal damage in the ipsilateral hemisphere only. Phosphorylated-Akt levels were decreased from 0.5 to 6 h post-HI, whereas the level of phosphorylated extracellular signal-related kinases (ERK)1/2 increased during this time frame. Hypoxia-inducible factor (HIF)-1α protein increased with a peak at 3 h after HI. mRNA expression for IL-β and tumor necrosis factor-α and -β started to increase at 6 h with a peak at 24 h post-HI. Expression of heat shock protein 70 was increased from 12 h after HI onwards in the ipsilateral hemisphere only. Surprisingly, HI changed the expression of cytokines, HIF1-α ,and P-Akt to the same extent in both the ipsi- as well as the contralateral hemisphere, although neuronal damage was unilateral. Exposure of animals to hypoxia without carotid artery occlusion induced similar changes in cytokines, HIF-1α, and P-Akt. We conclude that during HI, hypoxia is sufficient to regulate multiple molecular mediators that may contribute, but are not sufficient, to induce long-term neuronal damage.