Spinal Cord Injury (SCI) induces localized tissue damage that extends to neighboring cells and tissues within hours, days, or even weeks, exacerbating the severity of the injury. During the progressive aggravation of the injury, intracellular Nicotinamide adenine dinucleotide (NAD+) levels rapidly decline. NAD + is a crucial coenzyme involved in various physiological processes, particularly energy metabolism, and plays a vital role in tissue repair. Depletion of NAD+ adversely affects and amplifies tissue damage. Exogenous supplementation of NAD+ precursors and other means can increase NAD+ levels. Nicotinamide mononucleotide (NMN), a direct precursor of NAD+, has demonstrated safety and efficacy in enhancing NAD+ content and protecting tissues in preclinical and clinical studies. However, there is still limited evidence regarding the neuroprotective effects of NMN in spinal cord injury. In this study, we propose the hypothesis that NMN can elevate NAD+ levels, safeguard spinal cord tissues, attenuate post-injury inflammatory responses, and promote motor recovery. Our findings revealed that intraperitoneal injection of NMN (500 mg/kg) immediately after moderate contusion-type spinal cord injury and daily for 7 consecutive days significantly raised NAD+ levels in mouse spinal cord tissues. Histological evaluation using paraffin hematoxylin-eosin staining of spinal cord vacuole area and immunofluorescence staining of neuron count and fluorescence intensity demonstrated that NMN administration reduced the vacuole area in spinal cord tissues and increased the number of neurons, thereby safeguarding the spinal cord tissues. Improved motor function was indicated by BMS score, gait coordination analysis, grip strength assessment, and baton transfer endurance assessment. Transcriptome sequencing, qRT-PCR, protein microarray, and ELISA results collectively suggest that NMN may exert neuroprotective effects by reducing inflammatory responses. Furthermore, Western Blot analysis indicated that this effect may be mediated via the NF-κB pathway. In conclusion, these findings suggest that NMN, an NAD⁺ precursor, increases NAD⁺ levels in injured spinal cord tissue. NMN alleviates tissue injury and exerts neuroprotective effects, potentially suppressing neuroinflammation in an NF-κB pathway-dependent manner, thereby promoting locomotor recovery after SCI.
Exercise is widely recognized as an important modulator of hippocampal brain-derived neurotrophic factor (BDNF) and cognitive function; however, whether different exercise modalities, including endurance and motor skill training, produce distinct effects on hippocampal BDNF levels and cognitive performance remains unclear. Therefore, the present study investigated the effects of endurance training, rotarod training, and concurrent exercise on hippocampal BDNF levels and cognitive outcomes in male Wistar rats. Forty male Wistar rats were randomly assigned to endurance training (ET), rotarod training (RT), endurance followed by rotarod training (ERT), rotarod followed by endurance training (RET), or control (C) groups. After eight weeks of training, cognitive performance was evaluated using the Morris Water Maze (MWM), Novel Object Recognition (NOR), and passive avoidance tests to assess spatial learning and memory, recognition memory, and aversive memory, respectively. Hippocampal BDNF concentrations were measured using ELISA. Endurance training significantly increased hippocampal BDNF levels compared with the control, rotarod training (RT), and endurance-plus-rotarod (ERT) groups (p < 0.05). In addition, the rotarod-plus-endurance (RET) group exhibited higher BDNF concentrations than the RT group (p < 0.05). In the Morris Water Maze, training produced significant improvements in spatial learning across acquisition trials, with the RET group showing lower overall escape latency than the RT and control groups during acquisition (p < 0.05). In contrast, no significant differences were observed among groups in Novel Object Recognition or passive avoidance performance, and probe trial post hoc comparisons did not reveal significant between-group differences (p > 0.05). The present findings suggest that changes in hippocampal BDNF concentrations did not consistently parallel behavioral outcomes, indicating that BDNF responses and cognitive outcomes may vary across different exercise modalities. Not applicable.
Gut microbial alterations and brain metabolic disturbances have both been reported after cerebral ischemia, but their relationship with region-specific metabolic changes in the hippocampus remains unclear. This study characterized gut microbial and ipsilateral hippocampal metabolic alterations and evaluated hippocampal inflammatory and inflammasome-associated changes during the early subacute phase after permanent middle cerebral artery occlusion (MCAO). Male Sprague-Dawley rats were assigned to Naïve or permanent MCAO groups. On day 7, paired cecal content and ipsilateral hippocampal samples collected from the same rats were analyzed by 16 S rRNA gene sequencing and untargeted UPLC-MS/MS metabolomics (n = 8/group). Spearman correlation analysis was performed using matched microbiota and metabolomic profiles. In separate animal subsets, cerebral perfusion, infarction, hippocampal neuronal injury, inflammatory and eNOS-related markers, astrocytic reactivity, and inflammasome-associated proteins were assessed using laser speckle contrast imaging, TTC staining, histology, immunohistochemistry, immunofluorescence, and ELISA. GSE22255 was examined as contextual evidence of longer-term peripheral inflammatory gene expression after human ischemic stroke. Permanent MCAO was accompanied by reduced microbial richness and diversity and a distinct cecal microbial composition. The relative abundances of Escherichia-Shigella, Erysipelotrichaceae_UCG-003, and Parabacteroides were higher, whereas that of Bifidobacterium was lower, in MCAO rats. Hippocampal metabolomics revealed a lipid-centered metabolic phenotype characterized by increased signals putatively annotated as lysophosphatidylcholines, L-palmitoylcarnitine, butyrylcarnitine, and taurohyocholate, together with a decreased signal putatively annotated as homocarnosine. Pooled cross-omic analysis identified significant genus–metabolite associations, and the Erysipelotrichaceae_UCG-003–taurohyocholate association remained significant after adjustment for group membership. MCAO rats also exhibited reduced cerebral perfusion, cerebral infarction, hippocampal neuronal injury, increased TNF-α immunoreactivity and GFAP immunoreactivity, reduced eNOS immunoreactivity, and higher hippocampal levels of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18. Analysis of GSE22255 showed higher LBP, NLRP3, and IL1B expression in peripheral blood mononuclear cells collected at least 6 months after stroke. At day 7 after permanent MCAO, cecal microbiota remodeling and a lipid-related metabolic phenotype in the ipsilateral hippocampus were observed, with inflammatory and inflammasome-associated changes identified across separate experimental subsets. Paired cross-omic associations provide hypotheses for subsequent mechanistic investigation but do not establish a causal gut–brain relationship.
Single-nucleus RNA sequencing (snRNA-seq) is pivotal for dissecting cellular heterogeneity in the brain, yet the effects of technical variables, namely tissue harvest, nuclear fixation, and sequencing platform, on cell composition and gene expression remain poorly quantified. Here, we generated snRNA-seq data from adult mouse brains under distinct experimental designs to present a systematic multi-factorial benchmarking study. This work dissects the individual and interactive effects of harvest methods (post-mortem vs. in vivo), fixation treatments (methanol vs. fresh), and sequencing platforms (10x Genomics vs. SeekOne Digital Droplet). The standard post-mortem harvest induces rapid, selective loss of metabolically active glutamatergic neuron subpopulations, preserving global transcriptome but obscuring transcripts related to advanced cognitive functions. Methanol fixation minimally altered cell-type compositions but introduced cell-type-specific gene expression shifts, preferentially stabilizing structural and core functional transcripts in neurons and glia. Crucially, the sequencing platform shaped biological interpretation. The 10x and SeekOne platforms captured largely non-overlapping differential expression signatures in response to harvest stress, with 10x showing a bias toward detecting synaptic transcript degradation and SeekOne toward preserved metabolic and glial programs. Our study provides a systematic map of technical confounders in snRNA-seq of mouse brain, identifying glutamatergic subpopulations as particularly vulnerable and platform choice as a critical, non-neutral variable. It also provides an experimental and analytical framework to separate technical artifacts from true biological signal, which is important for building reproducible and integrative brain cell atlases. These findings suggest that precise reporting and control of workflow variables are prerequisites for accurate biological discovery in single-cell transcriptomics studies.
This preliminary study aimed to examine whether exercise-induced hypohydration is associated with different prefrontal hemodynamic and working-memory responses in male athletes with high and low body mass index (BMI). We hypothesized that athletes with high BMI would show less favorable prefrontal oxygenation patterns and reduced cognitive task performance following exercise-induced hypohydration. Twelve trained male athletes were divided into a high-BMI group (HBMIG; BMI = 33.91 ± 5.73 kg/m²) and a low-BMI group (LBMIG; BMI = 24.68 ± 2.54 kg/m²). Participants completed a familiarization session and an experimental session. During the experimental session, body mass and hematocrit were measured before and after exercise. Prefrontal hemodynamic responses were recorded using functional near-infrared spectroscopy during a 2-back working-memory task. The exercise protocol consisted of 30 min of constant-load cycling at 100 W and 60 rpm, followed by 4 × 20-s sprint intervals. Hematocrit levels increased in both groups compared to pre-exercise levels (p < 0.05), with a higher increase in HBMIG (p = 0.045). On the other hand, pre-post exercise responses in prefrontal hemodynamic activity during the 2-back test showed different patterns for each group: an increase in oxyhemoglobin for the LBMIG (p = 0.002), an increase in deoxyhemoglobin for the HBMIG (p = 0.033), and an increase in total hemoglobin for the HBMIG (p = 0.050). Furthermore, the number of missing answers in the post-exercise 2-back test results was significantly higher in the HBMIG compared to the LBMIG. These Preliminary findings suggest that male athletes with high BMI may exhibit different prefrontal hemodynamic responses and reduced task engagement or working-memory performance under exercise-induced hypohydration. However, given the small sample size and absence of a euhydrated control condition, these findings should be interpreted with caution and confirmed in larger controlled studies.
