Probable Novel APP Met671Leu Mutation in a Chinese Han Family with Early-Onset Alzheimer's Disease.

Familial Alzheimer's disease (AD) is a rare disease caused by autosomal-dominant mutations. APP (encoding amyloid precursor protein), PSEN1 (encoding presenilin 1), and PSEN2 (encoding presenilin 2) are the most common genes cause dominant inherited AD. This study aimed to demonstrate a Chinese early-onset AD pedigree presenting as progressive memory impairment, apraxia, visual-spatial disorders, psychobehavioral disorders, and personality changes with a novel APP gene mutation. The family contains four patients, three carries and three normal family members. The proband underwent brain magnetic resonance imaging (MRI), 18F-fludeoxyglucose positron emission tomography (18F-FDG-PET), cerebrospinal fluid amyloid detection, 18F-florbetapir (AV-45) Positron Emission Computed Tomography (PET) imaging, whole-exome sequencing and Sanger sequencing. Brain MRI images showed brain atrophy, especially in the entorhinal cortex, temporal hippocampus, and lateral ventricle dilation. The FDG-PET showed hypometabolism in the frontotemporal, parietal, and hippocampal regions. 18F-florbetapir (AV-45) PET imaging showed cerebral cortex Aß protein deposition. The cerebrospinal fluid amyloid protein test showed Aß42/Aß40 ratio decreases, pathological phosphor-tau level increases. Whole-exome sequencing detected a new missense mutation of codon 671 (M671L), which was a heterozygous A to T point mutation at position 2011 (c.2011A > T) in exon 16 of the amyloid precursor protein, resulting in the replacement of methionine to Leucine. The co-separation analysis was validated in this family. The mutation was found in 3 patients, 3 clinical normal members in the family, but not in the other 3 unaffected family members, 100 unrelated normal subjects, or 100 sporadic patients with AD. This mutation was probably pathogenic and novel in a Chinese Han family with early-onset AD.

C-kit controls blood-brain barrier permeability by regulating caveolae-mediated transcytosis after chronic cerebral hypoperfusion.

Blood-brain barrier (BBB) dysfunction plays a pivotal role in the pathology of chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases. Continuous endothelial cells (EC) that line the blood vessels of the brain are important components of the BBB to strictly control the flow of substances and maintain the homeostatic environment of the brain. However, the molecular mechanisms from the perspective of EC-induced BBB dysfunction after CCH are largely unknown. In this study, the BBB function was assessed using immunostaining and transmission electron microscopy. The EC dysfunction profile was screened by using EC enrichment followed by RNA sequencing. After identified the key EC dysfunction factor, C-kit, we used the C-kit inhibition drug (imatinib) and C-kit down-regulation method (AAV-BR1-C-kit shRNA) to verify the role of C-kit on BBB integrity and EC transcytosis after CCH. Furthermore, we also activated C-kit with stem cell factor (SCF) to observe the effects of C-kit on BBB following CCH. We explored that macromolecular proteins entered the brain mainly through EC transcytosis after CCH and caused neuronal loss. Additionally, we identified receptor tyrosine kinase C-kit as a key EC dysfunction molecule. Furthermore, the pharmacological inhibition of C-kit with imatinib counteracted BBB leakage by reducing caveolae-mediated transcytosis. Moreover, treatment with AAV-BR1-C-kit shRNA, which targets brain EC to inhibit C-kit expression, also ameliorated BBB leakage by reducing caveolae-mediated transcytosis. Furthermore, the SCF increased the permeability of the BBB by actively increasing caveolae-mediated transcytosis. This study provides evidence that C-kit is a key BBB permeability regulator through caveolae-mediated transcytosis in EC after CCH.

Presenilin2 D439A Mutation Induces Dysfunction of Mitochondrial Fusion/Fission Dynamics and Abnormal Regulation of GTPase Activity.

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease, and approximately 10% of AD cases are early-onset familial AD (EOFAD), which is mainly linked to point mutations in genes encoding presenilins (PS1 and PS2). Mutations in PS2 are extremely rare and have not received enough attention. Recently, studies have found that Rho GTPase activity is closely related to the pathogenesis of AD. In this study, we used transcriptome sequencing in PS2 siRNA-transfected SH-SY5Y cells and found a group of differentially expressed genes (DEGs) related to the regulation of GTPase activity. Among those DEGs, the most significantly downregulated was Rho guanine nucleotide exchange factor 5 (ARHGEF5). GTPase activity in PS2 siRNA-transfected cells was significantly decreased. Then, we found that the expression of ARHGEF5 and the GTPase activity of Mitochondrial Rho GTPase 2 (Miro2) in PS2 D439A mutant SH-SY5Y cells were significantly decreased. We found for the first time that PS2 can bind to Miro2, and the PS2 D439A mutation reduced the binding between PS2 and Miro2, reduced the expression of Miro2, and resulted in an imbalance in mitochondrial fusion/fission dynamics. In conclusion, PS2 gene knockdown may participate in the pathogenesis of AD through the regulation of GTPase activity. The imbalance in mitochondrial dynamics mediated by the PS2 D439A mutation through regulation of the expression and GTPase activity of Miro2 may be a potential pathogenic mechanism of AD.

A Chinese SCA36 pedigree analysis of NOP56 expansion region based on long-read sequencing.

Introduction: Spinocerebellar ataxias 36 (SCA36) is the neurodegenerative disease caused by the GGCCTG Hexanucleotide repeat expansions in NOP56, which is too long to sequence using short-read sequencing. Single molecule real time (SMRT) sequencing can sequence across disease-causing repeat expansion. We report the first long-read sequencing data across the expansion region in SCA36. Methods: We collected and described the clinical manifestations and imaging features of Han Chinese pedigree with three generations of SCA36. Also, we focused on structural variation analysis for intron 1 of the NOP56 gene by SMRT sequencing in the assembled genome. Results: The main clinical features of this pedigree are late-onset ataxia symptoms, with a presymptomatic presence of affective and sleep disorders. In addition, the results of SMRT sequencing showed the specific repeat expansion region and demonstrated that the region was not composed of single GGCCTG hexanucleotides and there were random interruptions. Discussion: We extended the phenotypic spectrum of SCA36. We applied SMRT sequencing to reveal the correlation between genotype and phenotype of SCA36. Our findings indicated that long-read sequencing is well suited to characterize known repeat expansion.

VCAM1 Drives Vascular Inflammation Leading to Continuous Cortical Neuronal Loss Following Chronic Cerebral Hypoperfusion.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) is associated with neuronal loss and blood-brain barrier (BBB) impairment in vascular dementia (VaD). However, the relationship and the molecular mechanisms between BBB dysfunction and neuronal loss remain elusive. OBJECTIVE: We explored the reasons for neuron loss following CCH. METHODS: Using permanent bilateral common carotid artery occlusion (2VO) rat model, we observed the pathological changes of cortical neurons and BBB in the sham group as well as rats 3d, 7d, 14d and 28d post 2VO. In order to further explore the factors influencing neuron loss following CCH with regard to cortical blood vessels, we extracted cortical brain microvessels at five time points for transcriptome sequencing. Finally, integrin receptor a4ß1 (VLA-4) inhibitor was injected into the tail vein, and cortical neuron loss was detected again. RESULTS: We found that cortical neuron loss following CCH is a continuous process, but damage to the BBB is acute and transient. Results of cortical microvessel transcriptome analysis showed that biological processes related to vascular inflammation mainly occurred in the chronic phase. Meanwhile, cell adhesion molecules, cytokine-cytokine receptor interaction were significantly changed at this phase. Among them, the adhesion molecule VCAM1 plays an important role. Using VLA-4 inhibitor to block VCAM1-VLA-4 interaction, cortical neuron damage was ameliorated at 14d post 2VO. CONCLUSION: Injury of the BBB may not be the main reason for persistent loss of cortical neurons following CCH. The continuous inflammatory response within blood vessels maybe an important factor in the continuous loss of cortical neurons following CCH.

Effects of different regional cerebral blood flow on white matter hyperintensity in CADASIL patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an early-onset inherited small vessel disease. Decreased cerebral blood flow (CBF) may contribute to white matter hyperintensity (WMH) severity in CADASIL, but more evidence is needed to support this hypothesis. This study comprised six patients with CADASIL who harbored mutations in the coding sequence of NOTCH3 and twelve age-matched neurologically healthy controls. We collected clinical and imaging data from patients with CADASIL and divided the brain into four regions: WMH, normal-appearing white matter (NAWM), gray matter (GM), and global brain. We analyzed the relationship between CBF of each region and the WMH volume. Compared with the control group, CBF was significantly decreased in all four regions in the CADASIL group. Lower CBF in these regions was correlated with higher WMH volume in CADASIL. CBF in the NAWM, GM and global regions was positively correlated with that in WMH region. However, after correction tests, only CBF in the WMH region but not in NAWM, GM and global regions was associated with WMH volume. Our findings suggest that CBF in the WMH region is an influencing factor of the WMH severity in CADASIL.

Mechanisms regulating cerebral hypoperfusion in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy.

Cerebral small vessel disease (CSVD) is a leading cause of stroke and dementia. As the most common type of inherited CSVD, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is characterized by the NOTCH3 gene mutation which leads to Notch3 ectodomain deposition and extracellular matrix aggregation around the small vessels. It further causes smooth muscle cell degeneration and small vessel arteriopathy in the central nervous system. Compromised cerebral blood flow occurs in the early stage of CADASIL and is associated with white matter hyperintensity, the typical neuroimaging pathology of CADASIL. This suggests that cerebral hypoperfusion may play an important role in the pathogenesis of CADASIL. However, the mechanistic linkage between NOTCH3 mutation and cerebral hypoperfusion remains unknown. Therefore, in this mini-review, it examines the cellular and molecular mechanisms contributing to cerebral hypoperfusion in CADASIL.

Novel PSEN1 (P284S) Mutation Causes Alzheimer's Disease with Cerebellar Amyloid ß-Protein Deposition.

BACKGROUND/OBJECTIVE: AD-associated PSEN1 mutations exhibit high clinical heterogeneity. The discovery of these mutations and the analysis of their associations with cases such as EOAD should be critical to understanding AD's pathogenesis. METHODS: We performed clinical analysis, neuroimaging, target region capture and high-throughput sequencing, and Sanger sequencing in a family of 3 generations. The underlying Alzheimer's pathology was evaluated using biomarker evidence obtained from cerebrospinal fluid (CSF) amyloid testing and 18F-florbetapir (AV-45) PET imaging. RESULTS: Target region capture sequencing revealed a novel heterozygous C to T missense point mutation at the base position 284 (c.850 C>T) located in exon 8 of the PSEN1 gene, resulting in a Prolineto- Serine substitution (P284S) at codon position 850. The mutation was also identified by Sanger sequencing in 2 family members, including proband and her daughter and was absent in the other 4 unaffected family members and 50 control subjects. Cerebrospinal fluid (CSF) amyloid test exhibited biomarker evidence of underlying Alzheimer's pathology. 18F-florbetapir (AV-45) PET imaging indicated extensive cerebral cortex and cerebellar Aß deposition. CONCLUSIONS: We discovered a novel PSEN1 pathogenic mutation, P284S, observed for the first time in a Chinese family with early-onset AD.

Chronic spinal cord injury repair by NT3-chitosan only occurs after clearance of the lesion scar.

Spinal cord injury (SCI) is a severe damage usually leading to limb dysesthesia, motor dysfunction, and other physiological disability. We have previously shown that NT3-chitosan could trigger an acute SCI repairment in rats and non-human primates. Due to the negative effect of inhibitory molecules in glial scar on axonal regeneration, however, the role of NT3-chitosan in the treatment of chronic SCI remains unclear. Compared with the fresh wound of acute SCI, how to handle the lesion core and glial scars is a major issue related to chronic-SCI repair. Here we report, in a chronic complete SCI rat model, establishment of magnetic resonance-diffusion tensor imaging (MR-DTI) methods to monitor spatial and temporal changes of the lesion area, which matched well with anatomical analyses. Clearance of the lesion core via suction of cystic tissues and trimming of solid scar tissues before introducing NT3-chitosan using either a rigid tubular scaffold or a soft gel form led to robust neural regeneration, which interconnected the severed ascending and descending axons and accompanied with electrophysiological and motor functional recovery. In contrast, cystic tissue extraction without scar trimming followed by NT3-chitosan injection, resulted in little, if any regeneration. Taken together, after lesion core clearance, NT3-chitosan can be used to enable chronic-SCI repair and MR-DTI-based mapping of lesion area and monitoring of ongoing regeneration can potentially be implemented in clinical studies for subacute/chronic-SCI repair.

Both the Complexity of Tight Junctions and Endothelial Transcytosis Are Increased During BBB Postnatal Development in Rats.

The blood-brain barrier (BBB) comprises a single layer of endothelial cells and maintains a safe and homeostatic environment for proper neuronal function and synaptic transmission. BBB is not a discrete physical barrier, but a complex, dynamic, and adaptable interface. BBB continues to mature under the influence of the neural environment within a short period of time after birth. However, the basic mechanism of BBB formation and maintenance remains a mystery. Early studies have identified two structural characteristics of microvascular endothelium: special tight junctions (TJs) and a very low transcellular vesicle transport rate. Previous studies believed that BBB damage was mainly due to the destruction of tight junctions, and the role of vesicle transcytosis was neglected, so there was a lack of research on its impact on blood-brain barrier. It is urgent to get a better clarification of the unique structural and functional characteristics of the BBB endothelium to explain the role of BBB injury in neurological diseases. RNA sequencing was used to study the molecular characterization of cerebral cortex vascular endothelium by isolating them from neonatal, adolescent and adult rats. For investigation the maintenance mechanism of the BBB, we focused on the cellular and molecular regulation of barrier formation and the two characteristics of microvascular endothelial cells. Interestingly, we found that during the development of the blood-brain barrier, although the tight junctions gradually mature, endothelial cell transcytosis is gradually enhanced, resulting in an increase in the permeability of the blood-brain barrier. This study suggested that under physiological conditions, low vesicle transport is playing an important role in maintaining the integrity of the blood-brain barrier. This study not only summarized the unique characteristics of microvascular endothelial cells, but also illustrated a clarified mechanism of the development and maintenance of BBB which can provide new therapeutic opportunities for central nervous system drug delivery. Raw data of RNA sequencing were deposited in NCBI Sequence Read Archive database (PRJNA790676).

Decreased mitochondrial D-loop region methylation mediates an increase in mitochondrial DNA copy number in CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a typical neurodegenerative disease associated with mitochondrial dysfunction. Methylation of the D-loop region and mitochondrial DNA copy number (mtDNAcn) play a critical role in the maintenance of mitochondrial function. However, the association between D-loop region methylation, mtDNAcn and CADASIL remains unclear. METHODS: Overall, 162 individuals were recruited, including 66 CADASIL patients and 96 age- and sex-matched controls. After extracting genomic DNA from the peripheral white blood cells, levels of D-loop methylation and mtDNAcn were assessed using MethylTarget sequencing and real-time PCR, respectively. RESULTS: We observed increased mtDNAcn and decreased D-loop methylation levels in CADASIL patients compared to the control group, regardless of gender stratification. Besides, we found a negative correlation between D-loop methylation levels and mtDNAcn. Mediation effect analysis shows that the proportion of the association between mtDNAcn and CADASIL that is mediated by D-loop methylation is 11.6% (95% CI 5.6, 22.6). After gender stratification, the proportions of such associations that are mediated by D-loop methylation in males and females were 7.2% (95% CI 2.4, 19.8) and 22.0% (95% CI 7.4, 50.1), respectively. CONCLUSION: Decreased methylation of the D-loop region mediates increased mtDNAcn in CADASIL, which may be caused by a compensatory mechanism of mitochondrial dysfunction in patients with CADASIL.

De novo Mutation Enables NOTCH3ECD Aggregation and Mitochondrial Dysfunction via Interactions with BAX and BCL-2.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 mutations is the most common monogenic hereditary pattern of cerebral small vessel disease. The aggregation of the mutant NOTCH3 may play a cytotoxic role in CADASIL. However, the main mechanism of this process remains unclear. OBJECTIVE: We aimed to investigate the possible pathogenesis of the mutant NOTCH3 in CADASIL. METHODS: The clinical information of two pedigrees were collected and analyzed. Furthermore, we constructed cell lines corresponding to this mutation in vitro. The degradation of the extracellular domain of NOTCH3 (NOTCH3ECD) was analyzed by Cycloheximide Pulse-Chase Experiment. Flow cytometry and cell counting kit-8 assay were performed to observe the effects of the NOTCH3 mutation on mitochondrial function and apoptosis. RESULTS: We confirmed a de novo heterozygous missense NOTCH3 mutation (c.1690G > A, p. A564T) in two pedigrees. In vitro, the NOTCH3ECD aggregation of A564T mutant may be related to their more difficult to degrade. The mitochondrial membrane potential was attenuated, and cell viability was significant decreased in NOTCH3ECD A564T group. Interestingly, BAX and cytochrome c were significantly increased, which are closely related to the mitochondrial-mediated pathway to apoptosis. CONCLUSION: In our study, the aggregation of NOTCH3ECD A564T mutation may be associated with more difficult degradation of the mutant, and the aggregation may produce toxic effects to induce apoptosis through the mitochondrial-mediated pathway. Therefore, we speculated that mitochondrial dysfunction may hopefully become a new breakthrough point to explain the pathogenesis of cysteine-sparing NOTCH3 mutations.

Genotype-phenotype correlations of heterozygous HTRA1-related cerebral small vessel disease: case report and systematic review.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is caused by biallelic HTRA1 pathogenic variants. Recent studies have shown that heterozygous HTRA1 mutations are associated with autosomal dominant cerebral small vessel disease (CSVD). However, large studies evaluating heterozygous HTRA1 carriers are lacking and the genotype-phenotype correlation is unknown. This study aimed to describe these mutations to clarify factors playing a role in the clinical phenotype amongst these patients. We reported two unrelated families and performed a systematic review of all published cases of heterozygous HTRA1-related CSVD. The clinical phenotype severity was independently related to the pathogenicity score (CADD score; p < 0.05) and mutation in the loop 3/loop D domains (p = 0.05); the pathogenicity score was also associated with exon distribution. More importantly, patients with mutations in exon 4 (p = 0.0001) or vascular risk factors (p < 0.05) presented with more severe clinical symptoms. Thus, clinical phenotype severity is influenced by the mutation domain and vascular risk factors. Applying the pathogenicity score to predict clinical outcomes and adopting preventive measures against cerebral vascular risk factors is advantageous.

Reduction in pericyte coverage leads to blood-brain barrier dysfunction via endothelial transcytosis following chronic cerebral hypoperfusion.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) is the leading cause of cerebral small vessel disease (CSVD). CCH is strongly associated with blood-brain barrier (BBB) dysfunction and white matter lesions (WMLs) in CSVD. However, the effects of CCH on BBB integrity and components and the cellular and molecular mechanisms underlying the effects of BBB dysfunction remain elusive. Whether maintaining BBB integrity can reverse CCH-induced brain damage has also not been explored. METHODS: In this study, we established a rat model of CSVD via permanent bilateral common carotid artery occlusion (2VO) to mimic the chronic hypoperfusive state of CSVD. The progression of BBB dysfunction and components of the BBB were assessed using immunostaining, Western blotting, transmission electron microscopy (TEM) and RNA sequencing. We also observed the protective role of imatinib, a tyrosine kinase inhibitor, on BBB integrity and neuroprotective function following CCH. The data were analyzed using one-way or two-way ANOVA. RESULTS: We noted transient yet severe breakdown of the BBB in the corpus callosum (CC) following CCH. The BBB was severely impaired as early as 1 day postoperation and most severely impaired 3 days postoperation. BBB breakdown preceded neuroinflammatory responses and the formation of WMLs. Moreover, pericyte loss was associated with BBB impairment, and the accumulation of serum protein was mediated by increased endothelial transcytosis in the CC. RNA sequencing also revealed increased transcytosis genes expression. BBB dysfunction led to brain damage through regulation of TGF-ß/Smad2 signaling. Furthermore, imatinib treatment ameliorated serum protein leakage, oligodendrocyte progenitor cell (OPC) activation, endothelial transcytosis, microglial activation, and aberrant TGF-ß/Smad2 signaling activation. CONCLUSIONS: Our results indicate that reduced pericyte coverage leads to increased BBB permeability via endothelial transcytosis. Imatinib executes a protective role on the BBB integrity via inhibition of endothelial transcytosis. Maintenance of BBB integrity ameliorates brain damage through regulation of TGF-ß/Smad2 signaling following CCH; therefore, reversal of BBB dysfunction may be a promising strategy for CSVD treatment.

Changes in the Morphology, Number, and Protein Levels of Plasma Exosomes in CADASIL Patients.

BACKGROUND: Exosomes are nano-sized extracellular vesicles which are secreted by cells and usually found in body fluids. Previous research has shown that exosomal secretion and autophagy-lysosomal pathway synergistically participates in intracellular abnormal protein elimination. The main pathological manifestations of Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is abnormal accumulation of mutant NOTCH3, and CADASIL vascular smooth muscle cells have been found with autophagy-lysosomal dysfunction. However, whether plasma exosomes change in CADASIL patients is still unclear. OBJECTIVE: We are aimed to investigate the differences of plasma exosomes between CADASIL patients and healthy controls. METHODS: The subjects included 30 CADASIL patients and 30 healthy controls without NOTCH3 mutation. The severity of white matter lesions (WMLs) of CADASIL patients was quantified by Fazekas score. Transmission electron microscopy and nanoparticle tracking analysis were performed to characterize plasma exosomes. In addition, NOTCH3, Neurofilament light and Aß42 levels in plasma exosomes were quantified by enzyme-linked immunosorbent assays. RESULTS: We found that exosomes from CADASIL patients were lower in quantity. In addition, CADASIL plasma exosomes had significantly lower levels of NOTCH3 and significantly increased levels of NFL than those of matched healthy subjects. Interestingly, plasma exosome NOTCH3 levels of CADASIL patients significantly correlated with severity of WMLs. CONCLUSION: The exosome NOTCH3 may be related to the pathological changes of CADASIL, which provides a basis for the pathogenesis research of CADASIL. In addition, plasma exosome NOTCH3 and NFL levels may act as biomarkers to monitor and predict disease progression and measure therapeutic effectiveness in the future clinical trials.

Differentiation of Bone Marrow Mesenchymal Stem Cells into Neural Lineage Cells Induced by bFGF-Chitosan Controlled Release System.

Bone marrow mesenchymal stem cells undergo differentiation to different lineages with different efficiencies when induced by different factors. We added a bFGF-chitosan controlled release system (bFGF-CCRS) as an inducer into conditioned medium to facilitate the oriented differentiation of BMSCs into neural lineage cells (eventually mature neurons); furthermore, we synchronized BMSCs to the G0/G1 phase via serum starvation to observe the effect of the inducer on the differentiation direction and efficiency. The nonsynchronized group, chitosan alone (not loaded with bFGF) group, soluble bFGF group, and conditioned medium group served as controls, and we observed the dynamic process of differentiation of BMSCs into neural lineage cells at different time points after the beginning of coculture. We analyzed the binding patterns of bFGF and chitosan and assayed the expression differences of key factors (FGFR1, ERK, and c-fos) and molecular switches (BTG2) that regulate the transformation from cell proliferation to differentiation. We also investigated the potential molecular mechanism of BMSC differentiation into neural lineage cells at a high percentage when induced by bFGF-CCRS.

bFGF-Sodium Hyaluronate Collagen Scaffolds Enable the Formation of Nascent Neural Networks After Adult Spinal Cord Injury.

Neural circuit reconstruction is the main target of functional restoration after adult spinal cord injury (SCI). The microenvironment after adult SCI is hostile to neural regeneration. Here, we designed a bFGF controlled releasing system (bFGF-CRS) by loading bFGF onto the sodium hyaluronate collagen scaffolds to modify the hostile microenvironment. We found that the bFGF-CRS scaffolds had proper mechanical properties for spinal cord regeneration and could slowly release bFGF for up to 6 weeks under the physiological condition. After implantation, the bFGF-CRS scaffolds could reduce microglial activation, promote revascularization, elicit endogenous neurogenesis and promote regrowth of transected axons. The endogenous mature newly born neurons could form synaptic-like connections with each other or with host neurons, including cortex neurons, brainstem neurons and spinal interneurons. The functional nascent neural networks between the lesion area and the host spinal cord were established. It eventually led to hindlimb locomotion recovery. Our study suggests that the bFGF-CRS scaffolds, modifying the microenvironment of the lesion area, can rebuild the damaged neural circuit, thus support great potential for SCI treatment in the clinical application.

Validation study of neurotrophin-3-releasing chitosan facilitation of neural tissue generation in the severely injured adult rat spinal cord.

It was previously reported that a tube holding chitosan carriers loaded with neurotrophin-3 (NT-3), after insertion into a 5 mm long transection gap in the adult rat spinal cord, triggered de novo neural tissue generation and functional recovery. Here, we report an effort to validate these findings using stringent blinding methodologies, which are crucial for robustness in reproducing biomedical studies. Radio frequency identification (RFID) chips were utilized to label rats that were randomly assigned into three experimental groups: transection with chitosan-NT-3 implant (C-NT3), transection only (T-controls), and laminectomy only (S-controls), blinding the experimenters to the treatments. Three months after surgery, animals only known by their RFID were functionally, electrophysiologically, and anatomically assessed. The data were then collected into the proper groups and statistically analyzed. Neural tissue with nestin-, Tuj1-, and NeuN-positive cells was found bridging the transection gap in C-NT3 rats, but not in T-controls. Motor- and somatosensory-evoked potentials were detected in C-NT3 rats and S-controls, but not in T-controls. Hind limb movement was significantly better in C-NT3 rats compared with T-controls. Our validation study indicates that C-NT3 implants facilitate neural tissue generation, at least in part, by eliciting endogenous neurogenesis. Our data support the use of C-NT3 implants for tissue remodeling in the injured spinal cord.

NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury.

Spinal cord injury (SCI) often leads to permanent loss of motor, sensory, and autonomic functions. We have previously shown that neurotrophin3 (NT3)-loaded chitosan biodegradable material allowed for prolonged slow release of NT3 for 14 weeks under physiological conditions. Here we report that NT3-loaded chitosan, when inserted into a 1-cm gap of hemisectioned and excised adult rhesus monkey thoracic spinal cord, elicited robust axonal regeneration. Labeling of cortical motor neurons indicated motor axons in the corticospinal tract not only entered the injury site within the biomaterial but also grew across the 1-cm-long lesion area and into the distal spinal cord. Through a combination of magnetic resonance diffusion tensor imaging, functional MRI, electrophysiology, and kinematics-based quantitative walking behavioral analyses, we demonstrated that NT3-chitosan enabled robust neural regeneration accompanied by motor and sensory functional recovery. Given that monkeys and humans share similar genetics and physiology, our method is likely translatable to human SCI repair.

Application of the sodium hyaluronate-CNTF scaffolds in repairing adult rat spinal cord injury and facilitating neural network formation.

The present study aimed to explore the potential of the sodium hyaluronate-CNTF (ciliary neurotrophic factor) scaffold in activating endogenous neurogenesis and facilitating neural network re-formation after the adult rat spinal cord injury (SCI). After completely cutting and removing a 5-mm adult rat T8 segment, a sodium hyaluronate-CNTF scaffold was implanted into the lesion area. Dil tracing and immunofluorescence staining were used to observe the proliferation, differentiation and integration of neural stem cells (NSCs) after SCI. A planar multielectrode dish system (MED64) was used to test the electrophysiological characteristics of the regenerated neural network in the lesioned area. Electrophysiology and behavior evaluation were used to evaluate functional recovery of paraplegic rat hindlimbs. The Dil tracing and immunofluorescence results suggest that the sodium hyaluronate-CNTF scaffold could activate the NSCs originating from the spinal cord ependymal, and facilitate their migration to the lesion area and differentiation into mature neurons, which were capable of forming synaptic contact and receiving glutamatergic excitatory synaptic input. The MED64 results suggest that functional synapsis could be established among regenerated neurons as well as between regenerated neurons and the host tissue, which has been evidenced to be glutamatergic excitatory synapsis. The electrophysiology and behavior evaluation results indicate that the paraplegic rats' sensory and motor functions were recovered in some degree. Collectively, this study may shed light on paraplegia treatment in clinics.