Erik J. Plautz

Control of cerebral ischemia with magnetic nanoparticles

The precise manipulation of microcirculation in mice can facilitate mechanistic studies of brain injury and repair after ischemia, but this manipulation remains a technical challenge, particularly in conscious mice. We developed a technology that uses micromagnets to induce aggregation of magnetic nanoparticles to reversibly occlude blood flow in microvessels. This allowed induction of ischemia in a specific cortical region of conscious mice of any postnatal age, including perinatal and neonatal stages, with precise spatiotemporal control but without surgical intervention of the skull or artery. When combined with longitudinal live-imaging approaches, this technology facilitated the discovery of a feature of the ischemic cascade: selective loss of smooth muscle cells in juveniles but not adults shortly after onset of ischemia and during blood reperfusion.

Preconditioning-induced CXCL12 upregulation minimizes leukocyte infiltration after stroke in ischemia-tolerant mice.

Repetitive hypoxic preconditioning creates long-lasting, endogenous protection in a mouse model of stroke, characterized by reductions in leukocyte–endothelial adherence, inflammation, and infarct volumes. The constitutively expressed chemokine CXCL12 can be upregulated by hypoxia and limits leukocyte entry into brain parenchyma during central nervous system inflammatory autoimmune disease. We therefore hypothesized that the sustained tolerance to stroke induced by repetitive hypoxic preconditioning is mediated, in part, by long-term CXCL12 upregulation at the blood–brain barrier (BBB). In male Swiss Webster mice, repetitive hypoxic preconditioning elevated cortical CXCL12 protein levels, and the number of cortical CXCL12+ microvessels, for at least two weeks after the last hypoxic exposure. Repetitive hypoxic preconditioning-treated mice maintained more CXCL12-positive vessels than untreated controls following transient focal stroke, despite cortical decreases in CXCL12 mRNA and protein. Continuous administration of the CXCL12 receptor (CXCR4) antagonist AMD3100 for two weeks following repetitive hypoxic preconditioning countered the increase in CXCL12-positive microvessels, both prior to and following stroke. AMD3100 blocked the protective post-stroke reductions in leukocyte diapedesis, including macrophages and NK cells, and blocked the protective effect of repetitive hypoxic preconditioning on lesion volume, but had no effect on blood–brain barrier dysfunction. These data suggest that CXCL12 upregulation prior to stroke onset, and its actions following stroke, contribute to the endogenous, anti-inflammatory phenotype induced by repetitive hypoxic preconditioning.

Effects of Subdural Monopolar Cortical Stimulation Paired With Rehabilitative Training on Behavioral and Neurophysiological Recovery After Cortical Ischemic Stroke in Adult Squirrel Monkeys.

Background. Cortical stimulation (CS) combined with rehabilitative training (RT) has proven effective for enhancing poststroke functional recovery in rats, but human clinical trials have had mixed outcomes. Objective. To assess the efficacy of CS/RT versus RT in a nonhuman primate model of cortical ischemic stroke. Methods. Squirrel monkeys learned a pellet retrieval task, then received an infarct to the distal forelimb (DFL) representation of primary motor cortex. A subdural monopolar electrode was implanted over the spared DFL representation in dorsal premotor cortex (PMD). Seven weeks postinfarct, monkeys underwent 4 to 6 weeks of RT (n = 8) or CS/RT (n = 7; 100 Hz, cathodal current) therapy. Behavioral performance was assessed before and after infarct, prior to therapy, and 1 and 12 weeks posttherapy (follow-up). The primary outcome measure was motor performance at 1 week posttherapy. Secondary outcomes included follow-up performance at 12 weeks and treatment-related changes in neurophysiological maps of spared DFL representations. Results. While postinfarct performance deficits were found in all monkeys, both groups demonstrated similar recovery profiles, with no difference in motor recovery between the RT and CS/RT groups. Posttherapy, PMD DFL area was significantly expanded in the RT group but not the CS/RT group. A significant relationship was found between motor recovery and DFL expansion in premotor cortex. Conclusions. Results suggest that the specific parameters utilized here were not optimal for promoting behavioral recovery in nonhuman primates. Though CS/RT has consistently shown efficacy in rat stroke models, the present finding has cautionary implications for translation of CS/RT therapy to clinical populations.

Involvement of aberrant cyclin-dependent kinase 5/p25 activity in experimental traumatic brain injury.

Traumatic brain injury (TBI) is associated with adverse effects on brain functions, including sensation, language, emotions and/or cognition. Therapies for improving outcomes following TBI are limited. A better understanding of the pathophysiological mechanisms of TBI may suggest novel treatment strategies to facilitate recovery and improve treatment outcome. Aberrant activation of cyclin‐dependent kinase 5 (Cdk5) has been implicated in neuronal injury and neurodegeneration. Cdk5 is a neuronal protein kinase activated via interaction with its cofactor p35 that regulates numerous neuronal functions, including synaptic remodeling and cognition. However, conversion of p35 to p25 via Ca2+‐dependent activation of calpain results in an aberrantly active Cdk5/p25 complex that is associated with neuronal damage and cell death. Here, we show that mice subjected to controlled cortical impact (CCI), a well‐established experimental TBI model, exhibit increased p25 levels and consistently elevated Cdk5‐dependent phosphorylation of microtubule‐associated protein tau and retinoblastoma (Rb) protein in hippocampal lysates. Moreover, CCI‐induced neuroinflammation as indicated by increased astrocytic activation and number of reactive microglia. Brain‐wide conditional Cdk5 knockout mice (Cdk5 cKO) subjected to CCI exhibited significantly reduced edema, ventricular dilation, and injury area. Finally, neurophysiological recordings revealed that CCI attenuated excitatory post‐synaptic potential field responses in the hippocampal CA3‐CA1 pathway 24 h after injury. This neurophysiological deficit was attenuated in Cdk5 cKO mice. Thus, TBI induces increased levels of p25 generation and aberrant Cdk5 activity, which contributes to pathophysiological processes underlying TBI progression. Hence, selectively preventing aberrant Cdk5 activity may be an effective acute strategy to improve recovery from TBI.

Physiologic Reelin does not play a strong role in protection against acute stroke

Stroke and Alzheimers disease, two diseases that disproportionately affect the aging population, share a subset of pathological findings and risk factors. The primary genetic risk factor after age for late-onset Alzheimers disease, ApoE4, has also been shown to increase stroke risk and the incidence of post-stroke dementia. One mechanism by which ApoE4 contributes to disease is by inducing in neurons a resistance to Reelin, a neuromodulator that enhances synaptic function. Previous studies in Reelin knockout mice suggest a role for Reelin in protection against stroke; however, these studies were limited by the developmental requirement for Reelin in neuronal migration. To address the question of the effect of Reelin loss on stroke susceptibility in an architecturally normal brain, we utilized a novel mouse with induced genetic reduction of Reelin. We found that after transient middle cerebral artery occlusion, mice with complete adult loss of Reelin exhibited a similar level of functional deficit and extent of infarct as control mice. Together, these results suggest that physiological Reelin does not play a strong role in protection against stroke pathology.

Mutant Screen Reveals the Piccolo\'s Control over Depression and Brain-Gonad Crosstalk

Successful sexual reproduction involves a highly complex, genetically encoded interplay between animal physiology and behavior. Here we developed a screen to identify genes essential for rat reproduction based on an unbiased methodology involving mutagenesis via the Sleeping Beauty transposon. As expected, our screen identified genes where reproductive failure was connected to gametogenesis (Btrc, Pan3, Spaca6, Ube2k) and embryogenesis (Alk3, Exoc6b, Slc1a3, Tmx4, Zmynd8). In addition, our screen identified Atg13 (longevity) Dlg1 and Pclo (neuronal disorders), previously not associated with reproduction. Dominant Pclo traits caused epileptiform activity and affected genes supporting GABAergic synaptic transmission (Gabra6, Gabrg3), and animals exhibited a compromised crosstalk between the brain and gonads via disturbed GnRH signaling. Recessive Pclo traits disrupted conspecific recognition required for courtship/mating and were mapped to allelic markers for major depressive disorder (Grm5, Htr2a, Sorcs3, Negr1, Drd2). Thus, Pclo-deficiency in rats link neural networks controlling sexual motivation to Pclo variants that have been associated with human neurological disorders.Successful sexual reproduction involves complex, genetically encoded interplay between animal physiology and behavior. The rat provides a highly fecund mammalian model for studying how the brain impacts reproduction. Here, we report a forward genetics screen in rats to identify genes that affect reproduction. A panel of 18 distinct rat strains harboring Sleeping Beauty gene trap mutations were analyzed for the ability to reproduce. As expected, our mutant screen identified genes where reproductive failure was connected to gametogenesis (Btrc, Pan3, Spaca6, Ube2k) and embryogenesis (Alk3, Exoc6b, Slc1a3, Tmx4, Zmynd8). In addition, we identified Atg13 (longevity) and Pclo (neuronal disorders), previously not associated with an inability to conceive. Neurologically, Pclo is known to regulate the size of presynaptic vesicle pools. Here, dominant traits in Pclo mutant rats caused epileptiform activity and affected genes supporting GABAergic synaptic transmission (Gabra6, Gabrg3). Recessive traits in Pclo mutant rats transmitted altered reproductive behavior, as homozygous Pclo mutant rats produced gametes but neither sex would mate with wildtype rats. Pclo mutant rat behavior was linked to endophenotypes signifying compromised brain-gonad crosstalk via disturbed GnRH signaling and allelic markers for major depressive disorder in humans (Grm5, Htr2a, Sorcs3, Negr1, Drd2). Thus, by rat genetics, we identified Pclo as a candidate presynaptic factor required for reproduction.Successful sexual reproduction involves complex, genetically encoded interplay between animal physiology and behavior. Here, we report an unbiased forward genetics screen to identify genes that regulate rat reproduction based on mutagenesis via the Sleeping Beauty transposon. As expected, our screen identified genes where reproductive failure was connected to gametogenesis (Btrc, Pan3, Spaca6, Ube2k) and embryogenesis (Alk3, Exoc6b, Slc1a3, Tmx4, Zmynd8). In addition, we identified Atg13 (longevity) and Pclo (neuronal disorders), previously not associated with an inability to conceive. Dominant Pclo traits caused epileptiform activity and affected genes supporting GABAergic synaptic transmission (Gabra6, Gabrg3). Recessive Pclo traits transmitted altered reproductive behavior, including reduced sexual motivation and increased aggression. Pclo mutant behavior was linked to hypothalamic markers for negative energy, compromised brain-gonad crosstalk via disturbed GnRH signaling and allelic markers for major depressive disorder (Grm5, Htr2a, Sorcs3, Negr1, Drd2). Thus, Pclo is a chemosensory-neuroendocrine regulatory factor that calibrates behavioral responses for reproduction.

Selective Non-nuclear Estrogen Receptor Activation Decreases Stroke Severity and Promotes Functional Recovery in Female Mice

Estrogens provide neuroprotection in animal models of stroke, but uterotrophic effects and cancer risk limit translation. Classic estrogen receptors (ERs) serve as transcription factors, whereas nonnuclear ERs govern numerous cell processes and exert beneficial cardiometabolic effects without uterine or breast cancer growth in mice. Here, we determined how nonnuclear ER stimulation with pathway-preferential estrogen (PaPE)-1 affects stroke outcome in mice. Ovariectomized female mice received vehicle, estradiol (E2), or PaPE-1 before and after transient middle cerebral artery occlusion (tMCAo). Lesion severity was assessed with MRI, and poststroke motor function was evaluated through 2 weeks after tMCAo. Circulating, spleen, and brain leukocyte subpopulations were quantified 3 days after tMCAo by flow cytometry, and neurogenesis and angiogenesis were evaluated histologically 2 weeks after tMCAo. Compared with vehicle, E2 and PaPE-1 reduced infarct volumes at 3 days after tMCAo, though only PaPE-1 reduced leukocyte infiltration into the ischemic brain. Unlike E2, PaPE-1 had no uterotrophic effect. Both interventions had negligible effect on long-term poststroke neuronal or vascular plasticity. All mice displayed a decline in motor performance at 2 days after tMCAo, and vehicle-treated mice did not improve thereafter. In contrast, E2 and PaPE-1 treatment afforded functional recovery at 6 days after tMCAo and beyond. Thus, the selective activation of nonnuclear ER by PaPE-1 decreased stroke severity and improved functional recovery in mice without undesirable uterotrophic effects. The beneficial effects of PaPE-1 are also associated with attenuated neuroinflammation in the brain. PaPE-1 and similar molecules may warrant consideration as efficacious ER modulators providing neuroprotection without detrimental effects on the uterus or cancer risk.