The development of thoracic and abdominal muscle depends on SDF1 and CXCR4.

In vertebrates, the lateral body wall muscle formation is thought to be initiated by direct outgrowth of the dermomyotomes resulting in the elongation of the hypaxial myotomes. This contrasts with the formation of the muscles of the girdle, limbs and intrinsic tongue muscles, which originate from long-range migrating progenitors. Previous work shows that the migration of these progenitors requires CXCR4 which is specifically expressed in the migrating cells, but not in the dermomyotome. Here, we show that cells in the ventrolateral-lip (VLL) of the dermomyotome at the flank level express CXCR4 in a pattern consistent with that of Pax3 and MyoR. In ovo gain-of-function experiments using electroporation of SDF-1 constructs into the VLL resulted in increased expression of c-Met, Pax3 and MyoD. In contrast, a loss-of-function approach by implantation of CXCR4-inhibitor beads into the VLL of the flank region caused a reduction in the expression of these markers. These data show that CXCR4 is expressed in the VLL, and by experimentally manipulating the CXCR4/SDF-1 signaling, we demonstrate the importance of this axis in body wall muscle development.

Multiple transcriptome analyses reveal mouse testis developmental dynamics.

The testes are the organs of gamete production and testosterone synthesis. Up to date, no model system is available for mammalian testicular development, and only few studies have characterized the mouse testis transcriptome from no more than three postnatal ages. To describe the transcriptome landscape of the developing mouse testis and identify the potential molecular mechanisms underlying testis maturation, we examined multiple RNA-seq data of mouse testes from 3-week-old (puberty) to 11-week-old (adult). Sperm cells appeared as expected in 5-week-old mouse testis, suggesting the proper sample collection. The principal components analysis revealed the genes from 3w to 4w clustered away from other timepoints, indicating they may be the important nodes for testicular development. The pairwise comparisons at two adjacent timepoints identified 7,612 differentially expressed genes (DEGs), resulting in 58 unique mRNA expression patterns. Enrichment analysis identified functions in tissue morphogenesis (3-4w), regulation of peptidase activity (4-5w), spermatogenesis (7-8w), and antigen processing (10-11w), suggesting distinct functions in different developmental periods. 50 hub genes and 10 gene cluster modules were identified in the testis maturation process by protein-protein interaction (PPI) network analysis, and the miRNA-lncRNA-mRNA, miRNA-circRNA-mRNA and miRNA-circRNA-lncRNA-mRNA competing endogenous RNA (ceRNA) networks were constructed. The results suggest that testis maturation is a complex developmental process modulated by various molecules, and that some potential RNA-RNA interactions may be involved in specific developmental stages. In summary, this study provides an update on the molecular basis of testis development, which may help to understand the molecular mechanisms of mouse testis development and provide guidance for mouse reproduction.

Adapting to new challenges in medical education: a three-step digitization approach for blended learning.

This research work focuses on evaluating the development of our three-step digitization approach, designed to transform traditional lectures into engaging digital learning experiences. Aimed at offering an easy-to-use solution for educators, this approach addresses the pressing challenges of modern educational environments by enhancing accessibility, engagement, and effectiveness of learning through digital means.Quantitative analysis demonstrated a notable increase in knowledge gain for students engaged with the digital format (t(230) = -2.795, p = 0.006) over traditional methods. Additionally, it was observed that the online setting significantly improved concentration levels (t(230) = -5.801, p < .001) and reduced distractions (t(230) = 2.848, p = 0.005). Emotional assessments, based on the Achievement Emotions Questionnaire (AEQ), indicated an elevation in enjoyment (t(230) = -4.717, p < .001) and a reduction in anxiety (t(230) = 9.446, p < .001) within the digital learning environment. A substantial preference for the digital course format was expressed by 61.0% of participants, with 71.4% acknowledging its superior efficiency compared to 14.3% for traditional lectures.Qualitative feedback underscored the digital format's flexibility, comprehensive learning experience, and immediate feedback as key benefits. Nevertheless, nuances such as a preferred understanding in face-to-face interactions and the absence of a social component in digital settings were noted.To conclude, the findings from this study illuminate the significant advantages of the three-step digitization approach in meeting contemporary educational challenges. By facilitating an enhanced knowledge acquisition and fostering a supportive emotional climate, this approach signifies a promising direction for the future of medical education and beyond, fusing the convenience of digital solutions with the depth and engagement of traditional learning methodologies.

Exploring the situational motivation of medical students through clinical medicine level test: a cross-sectional study.

China's Clinical Medicine Level Test (CMLT) was designed to evaluate the key factors that affect the learning outcome of medical students prior to entering clinical practice. In this study, we systemically analyzed the performance of a cohort of medical students at Jinan University School of Medicine participating in the recent CMLT. The analytical results of the medical students' written and objective structured clinical examination (OSCE) scores showed that their academic performance was predominantly associated with students' internship allocations, although other demographic characteristics such as sex, age, geographical origin of students, and grade point average (GPA) might be sporadically related to the students' OSCE performance at different OSCE stations. To explore the inherent reasons behind this, a survey was implemented among the medical students who participated in the examination to further interpret the factors influencing the students' learning outcome. The findings of this questionnaire manifested that intrinsic motivation and identified regulation acted as the major motivational profiles for the medical students from three different internship sites, whereas external regulation emerged as the crucial factor to make the students perform well academically in the CMLT. The result of this study suggested that strengthening the quality control of the clinical learning environment and improving medical administration may still be the most effective approaches to ensure the quality of clinical medical education.

atoh8 expression pattern in early zebrafish embryonic development.

Atonal homologue 8 (atoh8) is a basic helix-loop-helix transcription factor expressed in a variety of embryonic tissues. While several studies have implicated atoh8 in various developmental pathways in other species, its role in zebrafish development remains uncertain. So far, no studies have dealt with an in-depth in situ analysis of the tissue distribution of atoh8 in embryonic zebrafish. We set out to pinpoint the exact location of atoh8 expression in a detailed spatio-temporal analysis in zebrafish during the first 24 h of development (hpf). To our surprise, we observed transcription from pre-segmentation stages in the paraxial mesoderm and during the segmentation stages in the somitic sclerotome and not-as previously reported-in the myotome. With progressing maturation of the somites, the restriction of atoh8 to the sclerotomal compartment became evident. Double in situ hybridisation with atoh8 and myoD revealed that both genes are expressed in the somites at coinciding developmental stages; however, their domains do not spatially overlap. A second domain of atoh8 expression emerged in the embryonic brain in the developing cerebellum and hindbrain. Here, we observed a specific expression pattern which was again in contrast to the previously published suggestion of atoh8 transcription in neural crest cells. Our findings point towards a possible role of atoh8 in sclerotome, cerebellum and hindbrain development. More importantly, the results of this expression analysis provide new insights into early sclerotome development in zebrafish-a field of research in developmental biology which has not received much attention so far.

How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells.

Cell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18-HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

Dysbacteriosis-induced LPS elevation disturbs the development of muscle progenitor cells by interfering with retinoic acid signaling.

Whether myogenesis is affected by the maternal gut dysbacteriosis still remains ambiguous. In this study, first we show the elevated level of lipopolysaccharides (LPS) in a gut microbiota dysbiosis mouse model. Second, we demonstrate that the diameter of muscle fibers, limb development, and somitogenesis were inhibited in both the gut microbiota dysbiosis and LPS exposed mice and chicken embryos. These might be due to LPS disturbed the cell survival and key genes which regulate the somitogenesis and myogenesis. RNA sequencing and subsequent validation experiments verified that retinoic acid (RA) signaling perturbation was mainly responsible for the aberrant somite formation and differentiation. Subsequently, we found that LPS-induced reactive oxygen species (ROS generation and antioxidant genes such as Nrf2, AKR1B10) contributed to the above -mentioned interference with RA signaling. These findings highlight that the gut microbiota homeostasis is also involved in regulating the development of muscle progenitor cells during pregnancy.

Chicken Second Branchial Arch Progenitor Cells Contribute to Heart Musculature in vitro and in vivo.

In the past, the heart muscle was thought to originate from a single source of myocardial progenitor cells. More recently, however, an additional source of myocardial progenitors has been revealed to be the second heart field, and chicken embryos were important for establishing this concept. However, there have been few studies in chicken on how this field contributes to heart muscles in vitro. We have developed an ex vivo experimental system from chicken embryos between stages HH17-20 to investigate how mesodermal progenitors in the second branchial arch (BA2) differentiate into cardiac muscles. Using this method, we presented evidence that the progenitor cells within the BA2 arch differentiated into beating cardiomyocytes in vitro. The beating explant cells were positive for cardiac actin, Nkx2.5, and ventricular myosin heavy chain. In addition, we performed a time course for the expression of second heart field markers (Isl1 and Nkx2.5) in the BA2 from stage HH16 to stage HH21 using in situ hybridization. Accordingly, using EGFP-based cell labeling techniques and quail-chicken cell injection, we demonstrated that mesodermal cells from the BA2 contributed to the outflow tract and ventricular myocardium in vivo. Thus, our findings highlight the cardiogenic potential of chicken BA2 mesodermal cells in vitro and in vivo.

Zinc oxide nanoparticles exposure-induced oxidative stress restricts cranial neural crest development during chicken embryogenesis.

Zinc oxide Nanoparticles (ZnO NPs) are widely used as emerging materials in agricultural and food-related fields, which exists potential safety hazards to public health and environment while bringing an added level of convenience to our original life. It has been proved that ZnO NPs could be taken up by pregnant women and passed through human placental barrier. However, the toxic potential for embryo development remains largely unanswered. In this study, we discovered that ZnO NPs caused the cytotoxicity in vitro. Inhibition of free Zn2+ ions in solution by EDTA or inhibition of Zn2+ ions absorption by CaCl2 could partially eliminate ZnO NPs-mediated cell toxicity, though not redeem completely. This indicated that both nanoparticles and the release of Zn2+ ions were involved in ZnO NPs-mediated cytotoxicity. In addition, we also found that both nanoparticles and Zn2+ ion release triggered reactive oxygen species (ROS) production, which further induced cell toxicity, inflammation and apoptosis, which are mediated by NF-κB signaling cascades and the mitochondria dysfunction, respectively. Eventually, these events lead to the suppressed production and migration of cranial neural crest cells (CNCCs), which subsequently prompts the craniofacial defects in chicken embryos. The application of the antioxidant N-Acetyl-L-cysteine (NAC) rescued the ZnO NPs-induced cell toxicity and malformation of the CNCCs, which further verified our hypothesis. Our results revealed the relevant mechanism of ZnO NPs exposure-inhibited the development of CNCCs, which absolutely contribute to assess the risk of nanoparticles application.

Microbiota-derived lipopolysaccharide retards chondrocyte hypertrophy in the growth plate through elevating Sox9 expression.

Accumulating data show that the cytotoxicity of bacterial lipopolysaccharides (LPS) from microbiota or infection is associated with many disorders observed in the clinics. However, it is still obscure whether or not embryonic osteogenesis is affected by the LPS exposure during gestation. Using the early chicken embryo model, we could demonstrate that LPS exposure inhibits chondrogenesis of the 8-day chicken embryos by Alcian Blue-staining and osteogenesis of 17-day by Alcian Blue and Alizarin Red staining. Further analysis of the growth plates showed that the length of the proliferating zone (PZ) increases whereas that of the hypertrophic zone (HZ) decreased following LPS exposure. However there is no significant change on cell proliferation in the growth plates. Immunofluorescent staining, western blot analysis, and quantitive polymerase chain reaction revealed that Sox9 and Col2a1 are highly expressed at the messenger RNA level and their protein products are also abundant. LPS exposure causes a downregulation of Runx2 and Col10a1 expression in 8-day hindlimbs, and a suppression of Runx2, Col10a1, and Vegfa expression in 17-day phalanges. Knocking down Sox9 in ATDC5 cells by small interfering RNA transfection lead to the expression reduction of Col2a1, Runx2, and Col10a1, implying the vital role of Sox9 in the process of LPS-induced delay in the transition from proliferating chondrocytes to hypertrophic chondrocytes in the growth plate. In the presence of LPS, the antioxidant defense regulator nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is highly expressed, and the activities of superoxide dismutase 1 (SOD1), SOD2, and glutaredoxin rise in 17-day phalanges and ADTC5 cells. Simultaneously, an increase of intracellular ROS is observed. When Nrf2 expression was knocked down in ATDC5 cells, the expressions of Sox9, Col2a1, Runx2, Col10a1, and Vegfa were also going down as well. Taken together, our current data suggest that LPS exposure during gestation could restrict the chondrocytes conversion from proliferating to hypertrophic in the growth plate, in which LPS-induced Sox9 plays a crucial role to trigger the cascade of downstream genes by excessive ROS production and Nrf2 elevation.

Dexamethasone interferes with osteoblasts formation during osteogenesis through altering IGF-1-mediated angiogenesis.

Dexamethasone (Dex), a synthetic glucocorticoid (GC) with long-lasting treatment effects, has been proved to exert a modulatory effect on osteoblast proliferation and differentiation during embryonic osteogenesis. However, it is still controversial if Dex exposure influences endochondral ossification and the underlying mechanism. In this study, chick embryos in vivo and preosteoblast cell cultures in vitro were utilized to investigate the effects of Dex on osteoblast formation and differentiation during the skeletal development. We first demonstrated that Dex exposure could shorten the long bones of 17-day chick embryos in vivo, and also downregulated the expressions of osteogenesis-related genes. Next, we established that Dex exposure inhibited the proliferation and viability of preosteoblasts-MC3TC-E1 cells, and the addition of insulin-like growth factor 1 (IGF-1) could dramatically rescue these negative effects. On the basis of remarkable changes in the rescue experiments, we next verified the important role of angiogenesis in osteogenesis by culturing isolated embryonic phalanges in Dulbecco's modified Eagle's medium culture or on the chick chorioallantoic membrane (CAM). Then, we transplanted MC3T3-E1 cell masses onto the CAM. The data showed that Dex exposure reduced the vessel density within the developed cell mass, concomitantly with the downregulation of IGF-1 pathway. We verified that the inhibition of blood vessel formation caused by Dex could be rescued by IGF-1 treatment using the CAM angiogenesis model. Eventually, we demonstrated that the shortened length of the phalanges in the presence of Dex could be reversed by IGF-1 addition. In summary, these findings suggested that the inhibition of Igf-1 signal caused by Dex exposure exerts a detrimental impact on the formation of osteoblasts and angiogenesis, which consequently shortens long bones during osteogenesis.

Cell survival controlled by lens-derived Sema3A-Nrp1 is vital on caffeine-suppressed corneal innervation during chick organogenesis.

In this study, we investigated the effect of caffeine overexposure on corneal innervation in the early chicken embryo. Caffeine administration restricted corneal innervation by affecting trigeminal nerve development. Immunohistochemistry for phospho-Histone3 (pHIS3) and C-caspase3 revealed that cell survival was repressed by caffeine administration. Whole-mount in situ hybridization against semaphorin 3A (Sema3A) and neuropilin-1 (Nrp1) showed that both caffeine and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH, a free radical generator) administration upregulates the expression of both Sema3A and Nrp1. Next, we demonstrated that lens ablation in the developing chicken embryos significantly affected NF-labeled periocular nerve fascicles and innervation to the central eye region. Subsequently, we used a neuroblastoma cell line to investigate in vitro whether or not Sema3A-Nrp1 signaling exerts a key role on the caffeine-suppressed neuron survival. Knocking-down Sema3A through transfection with Sema3A-siRNA dramatically decreased the responsiveness of cells to caffeine administration, as well as cell apoptosis. We suggest that Sema3A-Nrp1 signaling regulates Trp53 and Cdkn1a through Slit2-Robo1 and Ephb2. Taken together, we speculate here that caffeine-enhanced reactive oxygen species upregulates Sema3A-Nrp1 expression in the lens and periocular tissues, resulting in corneal cell apoptosis, accompanied by its chemorepellent role on the invasion of the developing cornea by trigeminal sensory fibers.

The Hinrichsen Embryology Collection: Digitization of Historical Histological Human Embryonic Slides and MRI of Whole Fetuses.

The number of human embryology collections is very limited worldwide. Some of these comprise the Carnegie Collection, Kyoto Collection, and the Blechschmidt Collection. One further embryonic collection is the Hinrichsen Collection of the Ruhr University Bochum, Germany, which also contains very well-preserved embryos/fetuses, along with approximately 16,000 histological sections. The digitization of this collection is indispensable to enable conservation of the collection for the future and to provide a large group of embryologists, researchers, and physicians access to these histological slides. A small selection of these scans is available at the website of the Digital Embryology Consortium [https://-human-embryology.org/wiki/Main_Page].

The role of autophagy in morphogenesis and stem cell maintenance.

During embryonic development, cells need to undergo a number of morphological changes that are decisive for the shaping of the embryo's body, initiating organogenesis and differentiation into functional tissues. These remodeling processes are accompanied by profound changes in the cell membrane, the cytoskeleton, organelles, and extracellular matrix composition. While considerably detailed insight into the role of autophagy in stem cells biology has been gained in the recent years, information regarding the participation of autophagy in morphogenetic processes is only sparse. This review, therefore, focuses on the role of autophagy in cell morphogenesis through its regulatory activity in TGFß signaling, expression of adhesion molecules and cell cycle modification. It also discusses the role of autophagy in stem cell maintenance which is very fundamental for cell renewal and replacement during development, pathogenesis of certain diseases and development of therapies. We are thus addressing here perspectives for further potentially rewarding research topics.

Thymosin ß4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum.

Thymosin ß4 (Tß4), the principal G-actin regulating entity in eukaryotic cells, has also multiple intra- and extracellular functions related to tissue regeneration and healing. While its effect in adult organs is being widely investigated, currently, little is known about its influence on embryonic tissues, i.e., in the developing nervous system. The importance of Tß4 for neural stem cell proliferation in the embryonic chicken optic tectum (OT) has previously been shown by us for the first time. In the present study, using in ovo electroporation, we carried out a quantification of the effects of the Tß4-overexpression on the developing chicken OT between E4 and E6 at the hemisphere as well as cellular level. We precisely examined tissue growth and characterized cells arising from the elevated mitotic activity of progenitor cells. By using spinning-disk confocal laser scanning microscopy, we were able to visualize these effects across whole OT sections. Our experiments now demonstrate more clearly that the overexpression of Tß4 leads to a tangential expansion of the treated OT-hemisphere and that, under these circumstances, overall density of tectal and in particular of postmitotic neuronal cells is increased. Thanks to this new quantitative approach, the present results extend our previous findings that Tß4 is important for the proliferation of progenitor cells, neurogenesis, tangential expansion, and tissue growth in the young embryonic chicken optic tectum. Taken together, our results further illustrate and support the current idea that Tß4 is widely implicated in shaping and maintenance of the nervous system.

CREB, NF-Y and MEIS1 conserved binding sites are essential to balance Myostatin promoter/enhancer activity during early myogenesis.

Myostatin (MSTN) is a strong inhibitor of skeletal muscle growth in human and other vertebrates. Its transcription is controlled by a proximal promoter/enhancer (Mstn P/E) containing a TATA box besides CREB, NF-Y, MEIS1 and FXR transcription factor binding sites (TFBSs), which are conserved throughout evolution. The aim of this work was to investigate the role of these TFBSs on Mstn P/E activity and evaluate the potential of their putative ligands as Mstn trans regulators. Mstn P/E mutant constructs were used to establish the role of conserved TFBSs using dual-luciferase assays. Expression analyses were performed by RT-PCR and in situ hybridization in C2C12 myoblasts and E10.5 mouse embryos, respectively. Our results revealed that CREB, NF-Y and MEIS1 sites are required to balance Mstn P/E activity, keeping Mstn transcription within basal levels during myoblast proliferation. Furthermore, our data showed that NF-Y site is essential, although not sufficient, to mediate Mstn P/E transcriptional activity. In turn, CREB and MEIS1 binding sites seem to depend on the presence of NF-Y site to induce Mstn P/E. FXR appears not to confer any effect on Mstn P/E activity, except in the absence of all other conserved TFBS. Accordingly, expression studies pointed to CREB, NF-Y and MEIS1 but not to FXR factors as possible regulators of Mstn transcription in the myogenic context. Altogether, our findings indicated that CREB, NF-Y and MEIS1 conserved sites are essential to control basal Mstn transcription during early myogenesis, possibly by interacting with these or other related factors.

Development of the shoulder girdle musculature.

The muscles of the shoulder region are important for movements of the upper limbs and for stabilizing the girdle elements by connecting them to the trunk. They have a triple embryonic origin. First, the branchiomeric shoulder girdle muscles (sternocleidomastoideus and trapezius muscles) develop from the occipital lateral plate mesoderm using Tbx1 over the course of this development. The second population of cells constitutes the superficial shoulder girdle muscles (pectoral and latissimus dorsi muscles), which are derived from the wing premuscle mass. This muscle group undergoes a two-step development, referred to as the "in-out" mechanism. Myogenic precursor cells first migrate anterogradely into the wing bud. Subsequently, they migrate in a retrograde manner from the wing premuscle mass to the trunk. SDF-1/CXCR4 signaling is involved in this outward migration. A third group of shoulder muscles are the rhomboidei and serratus anterior muscles, which are referred to as deep shoulder girdle muscles; they are thought to be derived from the myotomes. It is, however, not clear how myotome cells make contact to the scapula to form these two muscles. In this review, we discuss the development of the shoulder girdle muscle in relation to the different muscle groups.

The development of anatomy: from macroscopic body dissections to stem cell-derived organoids.

Anatomy as a descriptive topic of research and instruction in medicine has been increasingly influenced by discoveries in molecular cell and developmental biology and most recently the advent of human induced pluripotent stem cells and organoids. We summarize here how anatomy has been influenced by developmental and stem cell biologists, and how in vitro modelling of the three-dimensional body environment is emerging to understand structure and function of cells during differentiation processes in development and disease.

A novel interaction between ATOH8 and PPP3CB.

ATOH8 is a bHLH transcription factor playing roles in a variety of developmental processes such as neurogenesis, differentiation of pancreatic precursor cells, development of kidney and muscle, and differentiation of endothelial cells. PPP3CB belongs to the catalytic subunit of the serine/threonine phosphatase, calcineurin, which can dephosphorylate its substrate proteins to regulate their physiological activities. In our study, we demonstrated that ATOH8 interacts with PPP3CB in vitro with different approaches. We show that the conserved catalytic domain of PPP3CB interacts with both the N-terminus and the bHLH domain of ATOH8. Although the interaction domain of PPP3CB is conserved among all isoforms of calcineurin A, ATOH8 selectively interacts with PPP3CB instead of PPP3CA, probably due to the unique proline-rich region present in the N-terminus of PPP3CB, which controls the specificity of its interaction partners. Furthermore, we show that inhibition of the interaction with calcineurin inhibitor, cyclosporin A (CsA), leads to the retention of ATOH8 to the cytoplasm, suggesting that the interaction renders nuclear localization of ATOH8 which may be critical to control its activity as transcription factor.

Inflammation and neuronal death in the motor cortex of the wobbler mouse, an ALS animal model.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder of the upper and lower motor neurons, characterized by rapid progressive weakness, muscle atrophy, dysarthria, dysphagia, and dyspnea. Whereas the exact cause of ALS remains uncertain, the wobbler mouse (phenotype WR; genotype wr/wr) equally develops a progressive degeneration of motor neurons in the spinal cord and motor cortex with striking similarities to sporadic human ALS, suggesting the possibility of a common pathway to cell death. METHODS: With the aid of immunohistochemistry, confocal laser scanning microscopy, and transmission electron microscopy techniques, we analyze the proliferation behavior of microglial cells and astrocytes. We also investigate possible motor neuron death in the mouse motor cortex at different stages of the wobbler disease, which so far has not received much attention. RESULTS: An abnormal density of Iba-1-positive microglial cells expressing pro-inflammatory tumor necrosis factor (TNF) alpha- and glial fibrillary acidic protein (GFAP)-positive activated astroglial cells was detected in the motor cortex region of the WR mouse 40 days postnatal (d.p.n.). Motor neurons in the same area show caspase 3 activation indicating neurodegenerative processes, which may cause progressive paralysis of the WR mice. It could also cause cell degeneration, such as vacuolization, dilation of the ER, and swollen mitochondria at the same time, and support the assumption that inflammation might be an important contributing factor of motor neuron degeneration. This would appear to be confirmed by the fact that there was no conspicuous increase of microglial cells and astrocytes in the motor cortex of control mice at any time. CONCLUSIONS: Activated microglial cells secrete a variety of pro-inflammatory and neurotoxic factors, such as TNF alpha, which could initiate apoptotic processes in the affected wobbler motor neurons, as reflected by caspase 3 activation, and thus, the neuroinflammatory processes might influence or exacerbate the neurodegeneration. Although it remains to be clarified whether the immune response is primary or secondary and how harmful or beneficial it is in the WR motor neuron disease, anti-inflammatory treatment might be considered.