Functional Motor Recovery from Motoneuron Axotomy Is Compromised in Mice with Defective Corticospinal Projections

Brachial plexus injury (BPI) and experimental spinal root avulsion result in loss of motor function in the affected segments. After root avulsion, significant motoneuron function is restored by re-implantation of the avulsed root. How much this functional recovery depends on corticospinal inputs is not known. Here, we studied that question using Celsr3|Emx1 mice, in which the corticospinal tract (CST) is genetically absent. In adult mice, we tore off right C5–C7 motor and sensory roots and re-implanted the right C6 roots. Behavioral studies showed impaired recovery of elbow flexion in Celsr3|Emx1 mice compared to controls. Five months after surgery, a reduced number of small axons, and higher G-ratio of inner to outer diameter of myelin sheaths were observed in mutant versus control mice. At early stages post-surgery, mutant mice displayed lower expression of GAP-43 in spinal cord and of myelin basic protein (MBP) in peripheral nerves than control animals. After five months, mutant animals had atrophy of the right biceps brachii, with less newly formed neuromuscular junctions (NMJs) and reduced peak-to-peak amplitudes in electromyogram (EMG), than controls. However, quite unexpectedly, a higher motoneuron survival rate was found in mutant than in control mice. Thus, following root avulsion/re-implantation, the absence of the CST is probably an important reason to hamper axonal regeneration and remyelination, as well as target re-innervation and formation of new NMJ, resulting in lower functional recovery, while fostering motoneuron survival. These results indicate that manipulation of corticospinal transmission may help improve functional recovery following BPI.     

Ocular Dominance Plasticity after Stroke Was Preserved in PSD-95 Knockout Mice

Neuronal plasticity is essential to enable rehabilitation when the brain suffers from injury, such as following a stroke. One of the most established models to study cortical plasticity is ocular dominance (OD) plasticity in the primary visual cortex (V1) of the mammalian brain induced by monocular deprivation (MD). We have previously shown that OD-plasticity in adult mouse V1 is absent after a photothrombotic (PT) stroke lesion in the adjacent primary somatosensory cortex (S1). Exposing lesioned mice to conditions which reduce the inhibitory tone in V1, such as raising animals in an enriched environment or short-term dark exposure, preserved OD-plasticity after an S1-lesion. Here we tested whether modification of excitatory circuits can also be beneficial for preserving V1-plasticity after stroke. Mice lacking postsynaptic density protein-95 (PSD-95), a signaling scaffold present at mature excitatory synapses, have lifelong juvenile-like OD-plasticity caused by an increased number of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) -silent synapses in V1 but unaltered inhibitory tone. In fact, using intrinsic signal optical imaging, we show here that OD-plasticity was preserved in V1 of adult PSD-95 KO mice after an S1-lesion but not in PSD-95 wildtype (WT)-mice. In addition, experience-enabled enhancement of the optomotor reflex of the open eye after MD was compromised in both lesioned PSD-95 KO and PSD-95 WT mice. Basic V1-activation and retinotopic map quality were, however, not different between lesioned PSD-95 KO mice and their WT littermates. The preserved OD-plasticity in the PSD-95 KO mice indicates that V1-plasticity after a distant stroke can be promoted by either changes in excitatory circuitry or by lowering the inhibitory tone in V1 as previously shown. Furthermore, the present data indicate that an increased number of AMPA-silent synapses preserves OD-plasticity not only in the healthy brain, but also in another experimental paradigm of cortical plasticity, namely the long-range influence on V1-plasticity after an S1-lesion.     

Restoring Cellular Energetics Promotes Axonal Regeneration and Functional Recovery after Spinal Cord Injury

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Stem Cell Mediation of Functional Recovery after Stroke in the Rat

Background

Regenerative strategies of stem cell grafting have been demonstrated to be effective in animal models of stroke. In those studies, the effectiveness of stem cells promoting functional recovery was assessed by behavioral testing. These behavioral studies do, however, not provide access to the understanding of the mechanisms underlying the observed functional outcome improvement.

Methodology/Principal Findings

In order to address the underlying mechanisms of stem cell mediated functional improvement, this functional improvement after stroke in the rat was investigated for six months after stroke by use of fMRI, somatosensory evoked potentials by electrophysiology, and sensorimotor behavior testing. Stem cells were grafted ipsilateral to the ischemic lesion. Rigorous exclusion of spontaneous recovery as confounding factor permitted to observe graft-related functional improvement beginning after 7 weeks and continuously increasing during the 6-month observation period. The major findings were i) functional improvement causally related to the stem cells grafting; ii) tissue replacement can be excluded as dominant factor for stem cell mediated functional improvement; iii) functional improvement occurs by exclusive restitution of the function in the original representation field, without clear contributions from reorganization processes, and iv) stem cells were not detectable any longer after six months.

Conclusions/Significance

A delayed functional improvement due to stem cell implantation has been documented by electrophysiology, fMRI and behavioral testing. This functional improvement occurred without cells acting as a tissue replacement for the necrotic tissue after the ischemic event. Combination of disappearance of grafted cells after six months on histological sections with persistent functional recovery was interpreted as paracrine effects by the grafted stem cells being the dominant mechanism of cell activity underlying the observed functional restitution of the original activation sites. Future studies will have to investigate whether the stem cell mediated improvement reactivates the original representation target field by using original connectivity pathways or by generating/activating new ones for the stimulus.     

Urokinase-Type Plasminogen Activator Deficiency Promotes Neoplasmatogenesis in the Colon of Mice

Urokinase-type plasminogen activator (uPA) participates in cancer-related biologic processes, such as wound healing and inflammation. The present study aimed to investigate the effect of uPA deficiency on the long-term outcome of early life episodes of dextran sodium sulfate (DSS)–induced colitis in mice. Wild-type (WT) and uPA-deficient (uPA^−/−) BALB/c mice were treated with DSS or remained untreated. Mice were necropsied either 1 week or 7 months after DSS treatment. Colon samples were analyzed by histopathology, immunohistochemistry, ELISA, and real-time polymerase chain reaction. At 7 months, with no colitis evident, half of the uPA^−/− mice had large colonic polypoid adenomas, whereas WT mice did not. One week after DSS treatment, there were typical DSS-induced colitis lesions in both WT and uPA^−/− mice. The affected colon of uPA^−/− mice, however, had features of delayed ulcer re-epithelialization and dysplastic lesions of higher grade developing on the basis of a significantly altered mucosal inflammatory milieu. The later was characterized by more neutrophils and macrophages, less regulatory T cells (Treg), significantly upregulated cytokines, including interleukin-6 (IL-6), IL-17, tumor necrosis factor-α, and IL-10, and lower levels of active transforming growth factor–β1 (TGF-β1) compared to WT mice. Dysfunctional Treg, more robust protumorigenic inflammatory events, and an inherited inability to produce adequate amounts of extracellular active TGF-β1 due to uPA deficiency are interlinked as probable explanations for the inflammatory-induced neoplasmatogenesis in the colon of uPA^−/− mice.     

Genetically-Defined Deficiency of Mannose-Binding Lectin Is Associated with Protection after Experimental Stroke in Mice and Outcome in Human Stroke

Background

The complement system is a major effector of innate immunity that has been involved in stroke brain damage. Complement activation occurs through the classical, alternative and lectin pathways. The latter is initiated by mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs). Here we investigated whether the lectin pathway contributes to stroke outcome in mice and humans.

Methodology/Principal Findings

Focal cerebral ischemia/reperfusion in MBL-null mice induced smaller infarctions, better functional outcome, and diminished C3 deposition and neutrophil infiltration than in wild-type mice. Accordingly, reconstitution of MBL-null mice with recombinant human MBL (rhMBL) enhanced brain damage. In order to investigate the clinical relevance of these experimental observations, a study of MBL2 and MASP-2 gene polymorphism rendering the lectin pathway dysfunctional was performed in 135 stroke patients. In logistic regression adjusted for age, gender and initial stroke severity, unfavourable outcome at 3 months was associated with MBL-sufficient genotype (OR 10.85, p = 0.008) and circulating MBL levels (OR 1.29, p = 0.04). Individuals carrying MBL-low genotypes (17.8%) had lower C3, C4, and CRP levels, and the proinflammatory cytokine profile was attenuated versus MBL-sufficient genotypes.

Conclusions/Significance

In conclusion, genetically defined MBL-deficiency is associated with a better outcome after acute stroke in mice and humans.     

Human Placenta-Derived Adherent Cell Treatment of Experimental Stroke Promotes Functional Recovery after Stroke in Young Adult and Older Rats

Background

Human Placenta-Derived Adherent Cells (PDAC®) are a novel mesenchymal-like cell population derived from normal human placental tissue. PDA-001 is a clinical formulation of PDAC® developed for intravenous administration. In this study, we investigated the efficacy of PDA-001 treatment in a rat model of transient middle cerebral artery occlusion (MCAo) in young adult (2–3 month old) and older rats (10–12 months old).

Methods

To evaluate efficacy and determine the optimal number of transplanted cells, young adult Wistar rats were subjected to MCAo and treated 1 day post MCAo with 1×10⁶, 4×10⁶ or 8×10⁶ PDA-001 cells (i.v.), vehicle or cell control. 4×10⁶ or 8×10⁶ PDA-001 cells were also tested in older rats after MCAo. Treatment response was evaluated using a battery of functional outcome tests, consisting of adhesive-removal test, modified Neurological Severity Score (mNSS) and foot-fault test. Young adult rats were sacrificed 56 days after MCAo, older rats were sacrificed 29 days after MCAo, and lesion volumes were measured using H&E. Immunohistochemical stainings for bromodeoxyuridine (BrdU) and von Willebrand Factor (vWF), and synaptophysin were performed.

Results

In young adult rats, treatment with 4×10⁶ PDA-001 cells significantly improved functional outcome after stroke (p<0.05). In older rats, significant functional improvement was observed with PDA-001 cell therapy in both of the 4×10⁶ and 8×10⁶ treatment groups. Functional benefits in young adult and older rats were associated with significant increases in the number of BrdU immunoreactive endothelial cells, vascular density and perimeter in the ischemic brain, as well as significantly increased synaptophysin expression in the ischemic border zone (p<0.05).

Conclusion

PDA-001 treatment significantly improved functional outcome after stroke in both young adult and older rats. The neurorestorative effects induced by PDA-001 treatment may be related to increased vascular density and synaptic plasticity.     

Increased Stathmin1 Expression in the Dentate Gyrus of Mice Causes Abnormal Axonal Arborizations

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3) were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.     

Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis

Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.     

Reduced Functional Connectivity of Default Mode and Set-Maintenance Networks in Ornithine Transcarbamylase Deficiency

Background and Purpose

Ornithine transcarbamylase deficiency (OTCD) is an X-chromosome linked urea cycle disorder (UCD) that causes hyperammonemic episodes leading to white matter injury and impairments in executive functioning, working memory, and motor planning. This study aims to investigate differences in functional connectivity of two resting-state networks—default mode and set-maintenance—between OTCD patients and healthy controls.

Methods

Sixteen patients with partial OTCD and twenty-two control participants underwent a resting-state scan using 3T fMRI. Combining independent component analysis (ICA) and region-of-interest (ROI) analyses, we identified the nodes that comprised each network in each group, and assessed internodal connectivity.

Results

Group comparisons revealed reduced functional connectivity in the default mode network (DMN) of OTCD patients, particularly between the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) node and bilateral inferior parietal lobule (IPL), as well as between the ACC/mPFC node and the posterior cingulate cortex (PCC) node. Patients also showed reduced connectivity in the set-maintenance network, especially between right anterior insula/frontal operculum (aI/fO) node and bilateral superior frontal gyrus (SFG), as well as between the right aI/fO and ACC and between the ACC and right SFG.

Conclusion

Internodal functional connectivity in the DMN and set-maintenance network is reduced in patients with partial OTCD compared to controls, most likely due to hyperammonemia-related white matter damage. Because several of the affected areas are involved in executive functioning, it is postulated that this reduced connectivity is an underlying cause of the deficits OTCD patients display in this cognitive domain.     

随机限食与重喂食小鼠的能量代谢和行为的可塑性

分别测定了随机限食和重喂食驯化的雌性KM小鼠的体重、摄食量、基础代谢率(BMR)、行为活动、身体脂肪和性腺重量.随机限食使摄食量增加、BMR和活动行为降低,生长发育迟缓,但对身体脂肪无显著影响.重喂食后上述指标均恢复到对照组水平,表现出显著的可塑性变化.结果表明,动物通过能量摄入和支出的权衡策略适应难以预测的食物资源变化,能量代谢和活动行为的可塑性调节在能量代谢的权衡策略中发挥重要作用.     

Clemastine Enhances Myelination,Delays Axonal Loss and Promotes Functional Recovery in Spinal Cord Injury

Recent evidence has shown that demyelination occurs along with axonal degeneration in spinal cord injury (SCI) during the secondary injury phase. Oligodendrocyte precursor cells (OPC) are present in the lesions but fail to differentiate into mature oligodendrocytes and form new myelin. Given the limited recovery of neuronal functions after SCI in adults without effective treatment available so far, it remains unknown whether enhancing OPC differentiation and myelination could benefit the recovery of SCI. To show the significance of myelin regeneration after SCI, the injury was treated with clemastine in the rat model. Clemastine is an FDA-approved drug that is potent in promoting oligodendrocyte differentiation and myelination in vivo, for four weeks following SCI. Motor function was assessed using sloping boards and grid walking tests and scored according to the Basso, Beattie, and Bresnahan protocol. The myelin integrity and protein expression were evaluated using transmission electron microscopy and immunofluorescence, respectively. The results indicated that clemastine treatment preserves myelin integrity, decreases loss of axons and improves functional recovery in the rat SCI model. The presented data suggest that myelination-enhancing strategies may serve as a potential therapeutic approach for the functional recovery in SCI.

     

Local Delivery of High-Dose Chondroitinase ABC in the Sub-Acute Stage Promotes Axonal Outgrowth and Functional Recovery after Complete Spinal Cord Transection

Chondroitin sulfate proteoglycans (CSPGs) are glial scar-associated molecules considered axonal regeneration inhibitors and can be digested by chondroitinase ABC (ChABC) to promote axonal regeneration after spinal cord injury (SCI). We previously demonstrated that intrathecal delivery of low-dose ChABC (1 U) in the acute stage of SCI promoted axonal regrowth and functional recovery. In this study, high-dose ChABC (50 U) introduced via intrathecal delivery induced subarachnoid hemorrhage and death within 48 h. However, most SCI patients are treated in the sub-acute or chronic stages, when the dense glial scar has formed and is minimally digested by intrathecal delivery of ChABC at the injury site. The present study investigated whether intraparenchymal delivery of ChABC in the sub-acute stage of complete spinal cord transection would promote axonal outgrowth and improve functional recovery. We observed no functional recovery following the low-dose ChABC (1 U or 5 U) treatments. Furthermore, animals treated with high-dose ChABC (50 U or 100 U) showed decreased CSPGs levels. The extent and area of the lesion were also dramatically decreased after ChABC treatment. The outgrowth of the regenerating axons was significantly increased, and some partially crossed the lesion site in the ChABC-treated groups. In addition, retrograde Fluoro-Gold (FG) labeling showed that the outgrowing axons could cross the lesion site and reach several brain stem nuclei involved in sensory and motor functions. The Basso, Beattie and Bresnahan (BBB) open field locomotor scores revealed that the ChABC treatment significantly improved functional recovery compared to the control group at eight weeks after treatment. Our study demonstrates that high-dose ChABC treatment in the sub-acute stage of SCI effectively improves glial scar digestion by reducing the lesion size and increasing axonal regrowth to the related functional nuclei, which promotes locomotor recovery. Thus, our results will aid in the treatment of spinal cord injury.     

Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

MUC5AC is the most abundant gel-forming mucin in the ocular system. However, the specific function is unknown. In the present study, a Muc5ac knockout (KO) mouse model was subject to various physiological measurements as compared to its wide-type (WT) control. Interestingly, when KO mice were compared to WT mice, the mean tear break up time (TBUT) values were significantly lower and corneal fluorescein staining scores were significantly higher. But the tear volume was not changed. Despite the lack of Muc5ac expression in the conjunctiva of KO mice, Muc5b expression was significantly increased in these mice. Corneal opacification, varying in location and severity, was found in a few KO mice but not in WT mice. The present results suggest a significant difference in the quality, but not the quantity, of tear fluid in the KO mice compared to WT mice. Dry eye disease is multifactorial and therefore further evaluation of the varying components of the tear film, lacrimal unit and corneal structure of these KO mice may help elucidate the role of mucins in dry eye disease. Because Muc5ac knockout mice have clinical features of dry eye, this mouse model will be extremely useful for further studies regarding the pathophysiology of the ocular surface in dry eye in humans.     

Spontaneous Reperfusion after In Situ Thromboembolic Stroke in Mice

Injection of thrombin into the middle cerebral artery (MCA) of mice has been proposed as a new model of thromboembolic stroke. The present study used sequential multiparametric Magnetic Resonance Imaging (MRI), including Magnetic Resonance Angiography (MRA), Diffusion-Weighted Imaging (DWI) and Perfusion-Weighted Imaging (PWI), to document MCA occlusion, PWI-DWI mismatch, and lesion development. In the first experiment, complete MCA occlusion and reproducible hypoperfusion were obtained in 85% of animals during the first hour after stroke onset. In the second experiment, 80% of animals showed partial to complete reperfusion during a three-hour follow-up. Spontaneous reperfusion thus contributed to the variability in ischemic volume in this model. The study confirmed the value of the model for evaluating new thrombolytic treatments, but calls for extended MRI follow-up at the acute stage in therapeutic studies.     

Therapeutic Effects of hMAPC and hMSC Transplantation after Stroke in Mice

Stroke represents an attractive target for stem cell therapy. Although different types of cells have been employed in animal models, a direct comparison between cell sources has not been performed. The aim of our study was to assess the effect of human multipotent adult progenitor cells (hMAPCs) and human mesenchymal stem cells (hMSCs) on endogenous neurogenesis, angiogenesis and inflammation following stroke. BALB/Ca-RAG 2(-/-) γC(-/-) mice subjected to FeCl(3) thrombosis mediated stroke were intracranially injected with 2×10(5) hMAPCs or hMSCs 2 days after stroke and followed for up to 28 days. We could not detect long-term engraftment of either cell population. However, in comparison with PBS-treated animals, hMSC and hMAPC grafted animals demonstrated significantly decreased loss of brain tissue. This was associated with increased angiogenesis, diminished inflammation and a glial-scar inhibitory effect. Moreover, enhanced proliferation of cells in the subventricular zone (SVZ) and survival of newly generated neuroblasts was observed. Interestingly, these neuroprotective effects were more pronounced in the group of animals treated with hMAPCs in comparison with hMSCs. Our results establish cell therapy with hMAPCs and hMSCs as a promising strategy for the treatment of stroke.     

Local Administration of AAV-BDNF to Subventricular Zone Induces Functional Recovery in Stroke Rats

Migration of new neuroprogenitor cells (NPCs) from the subventricular zone (SVZ) plays an important role in neurorepair after injury. Previous studies have shown that brain derived neurotrophic factor (BDNF) enhances the migration of NPCs from SVZ explants in neonatal mice in vitro. The purpose of this study was to identify the role of BDNF in SVZ cells using AAV-BDNF in an animal model of stroke. BDNF protein production after AAV‐BDNF infection was verified in primary neuronal culture. AAV-BDNF or AAV-RFP was injected into the left SVZ region of adult rats at 14 days prior to right middle cerebral artery occlusion (MCAo). SVZ tissues were collected from the brain and placed in Metrigel cultures 1 day after MCAo. Treatment with AAV-BDNF significantly increased the migration of SVZ cells in the stroke brain in vitro. In another set of animals, AAV-GFP was co-injected with AAV-BDNF or AAV-RFP to label cells in left SVZ prior to right MCAo. Local administration of AAV-BDNF significantly enhanced recovery of locomotor function and migration of GFP-positive cells from the SVZ toward the lesioned hemisphere in stroke rats. Our data suggest that focal administration of AAV-BDNF to the SVZ increases behavioral recovery post stroke, possibly through the enhancement of migration of cells from SVZ in stroke animals. Regional manipulation of BDNF expression through AAV may be a novel approach for neurorepair in stroke brains.     

Enhanced Proteostasis in Post-ischemic Stroke Mouse Brains by Ubiquilin-1 Promotes Functional Recovery

Stroke is pathologically associated with oxidative stress, protein damage, and neuronal loss. We previously reported that overexpression of a ubiquitin-like protein, ubiquilin-1 (Ubqln), protects neurons against ischemia-caused brain injury, while knockout of the gene exacerbates cerebral ischemia-caused neuronal damage and delays functional recovery. Although these observations indicate that Ubqln is a potential therapeutic target, transgenic manipulation-caused overexpression of Ubqln occurs before the event of ischemic stroke, and it remains unknown whether delayed Ubqln overexpression in post-ischemic brains within a clinically relevant time frame is still beneficial. To address this question, we generated lentiviruses (LVs) either overexpressing or knocking down mouse Ubqln, and treated post-ischemic stroke mice 6 h following the middle cerebral artery occlusion with the LVs before animal behaviors were evaluated at day 1, 3, 5, and 7. Our data indicate that post-ischemic overexpression of Ubqln significantly promoted functional recovery, whereas post-ischemic downregulation of Ubqln expression delays functional recovery. To further understand the mechanisms underlying how Ubqln functions, we also isolated protein aggregates from the brains of wild-type mice or the mice overexpressing Ubqln following ischemia/reperfusion. Western blot analysis indicates that overexpression of Ubqln significantly reduced the accumulation of protein aggregates. These observations not only suggest that Ubqln is a useful candidate for therapeutic intervention for ischemic stroke but also highlight the significance of proteostasis in functional recovery following stroke.