Ultrastructural analysis of collagen fibril diameter distribution in cleft lip

Objective: A preliminary study to determine collagen fibril diameter (CF-ED) distribution on medial and lateral sides of cleft lip (CL). Material and Methods: Tissue samples from medial and lateral sides of CL were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in Araldite CY212 resin for transmission electron microscopy. The analysis of CF-ED was performed using the ImageJ program. To characterize the packaging of collagen fibrils (CFs) in the two tissues, we estimated the collagen number density (CF-ND) and fibril-area-fraction (FAF). Differences in measurements across the two sides were calculated using Wilcoxon signed-rank test. Results: The CF-ED was statistically significantly (p < 0.001) smaller on the medial side (45.69 ± 7.89 nm) than on the lateral side (54.18 ± 7.62 nm). The medial side had a higher CF-ND and a higher percentage of FAF than the lateral side. Conclusion: Our finding of a smaller CF-ED and higher CF-ND and FAF for the medial side suggests possible differences in size and distribution of CFs between medial and lateral sides of CL. This finding provides knowledge toward underlying tissue biomechanics that may help reconstruction of perioral tissue scaffolds, ultimately resulting in better treatment of patients with oral clefts

Febuxostat Modulates MAPK/NF- κ

Xanthine oxidase and xanthine dehydrogenase have been implicated in producing myocardial damage following reperfusion of an occluded coronary artery. We investigated and compared the effect of febuxostat and allopurinol in an experimental model of ischemia-reperfusion (IR) injury with a focus on the signaling pathways involved. Male Wistar rats were orally administered vehicle (CMC) once daily (sham and IR + control), febuxostat (10 mg/kg/day; FEB10 + IR), or allopurinol (100 mg/kg/day; ALL100 + IR) for 14 days. On the 15th day, the IR-control and treatment groups were subjected to one-stage left anterior descending (LAD) coronary artery ligation for 45 minutes followed by a 60-minute reperfusion. Febuxostat and allopurinol pretreatment significantly improved cardiac function and maintained morphological alterations. They also attenuated oxidative stress and apoptosis by suppressing the expression of proapoptotic proteins (Bax and caspase-3), reducing TUNEL-positive cells, and increasing the level of antiapoptotic proteins (Bcl-2). The MAPK-based molecular mechanism revealed suppression of active JNK and p38 proteins concomitant with the rise in ERK1/ERK2, a prosurvival kinase. Additionally, a reduction in the level of inflammatory markers (TNF-α, IL-6, and NF-κB) was also observed. The changes observed with febuxostat were remarkable in comparison with those observed with allopurinol. Febuxostat protects relatively better against IR injury than allopurinol by suppressing inflammation and apoptosis mediating the MAPK/NF-κBp65/TNF-α pathway

Calbindin D-28K and parvalbumin expression in embryonic chick hippocampus is enhanced by prenatal auditory stimulation

Calcium-binding proteins (CaBPs) buffer excess of cytosolic Ca2+, which accompanies neuronal activity following external stimuli. Prenatal auditory stimulation by species-specific sound and music influences early maturation of the auditory pathway and the behavioral responses in chicks. In this study, we determined the volume, total number of neurons, proportion of calbindin D-28K and parvalbumin-positive neurons along with their levels of expression in the developing chick hippocampus following prenatal auditory stimulation. Fertilized eggs of domestic chicks were exposed to sounds of either species-specific calls or sitar music at 65 dB for 15 min/h round the clock from embryonic day (E) 10 until hatching. Hippocampi of developmental stages (E12, E16 and E20) were examined. With an increase in embryonic age during normal development, the hippocampus showed an increase in its volume, total number of neurons as well as in the neuron proportions and levels of expression of calbindin D-28K and parvalbumin. A significant increase of volume at E20 was noted only in the music-stimulated group compared to that of their age-matched control (p &lt; 0.05). On the other hand, both auditory-stimulated groups showed a significant increase in the proportion of immunopositive neurons and the levels of expression of calbindin D-28K and parvalbumin as compared to the control at all developmental stages studied (p &lt; 0.003). The increase in proportions of CaBP neurons during development and in the sound-enriched groups suggests an activity-dependent increase in Ca2+ influx. The enhanced expression of CaBPs may help in cell survival by preventing excitotoxic death of neurons during development and may also be involved in long-term potentiation

Prenatal acoustic stimulation influences neuronal size and the expression of calcium-binding proteins (calbindin D-28K and parvalbumin) in chick hippocampus

Prenatal auditory enrichment by species-specific sounds and sitar music enhances the expression of immediate early genes, synaptic proteins and calcium binding proteins (CaBPs) as well as modifies the structural components of the brainstem auditory nuclei and auditory imprinting area in chicks. There is also facilitation of postnatal auditory preference of the chicks to maternal calls following both types of sound stimulation indicating prenatal perceptual learning. To examine whether the sound enrichment protocol also affects the areas related to learning and memory, we assessed morphological changes in the hippocampus at post-hatch day 1 of control and prenatally sound-stimulated chicks. Additionally, the proportions of neurons containing calbindin D-28K and parvalbumin immunoreactivity as well as their protein levels were determined. Fertilized eggs of domestic chick were incubated under normal conditions of temperature, humidity, forced draft of air as well as light and dark (12:12 h) photoperiods. They were exposed to patterned sounds of species-specific and sitar music at 65 dB for 15 min per hour over a day/night cycle from day 10 of incubation till hatching. The hippocampal volume, neuronal nuclear size and total number of neurons showed a significant increase in the music-stimulated group as compared to the species-specific sound-stimulated and control groups. However, in both the auditory-stimulated groups the protein levels of calbindin and parvalbumin as well as the percentage of the immunopositive neurons were increased. The enhanced proportion of CaBPs in the sound-enriched groups suggests greater Ca2+ influx, which may influence long-term potentiation and short-term memory

Plasminogen activator inhibitor-1 promoter sequence variations in idiopathic osteonecrosis of head of femur

Background & objectives: Primary or idiopathic osteonecrosis of femur head (ONFH) is the second most commonly observed cause among Indian patients suffering from ischemic ONFH. Although a number of genetic polymorphisms have been associated with idiopathic ONFH pathogenesis in Korean and Chinese populations, there are no studies in the Indian population. This is an exploratory study designed to implicate in promoter sequence polymorphisms of a critical fibrinolytic system regulator, plasminogen activator inhibitor-1 (PAI-1) gene, in cases of idiopathic osteonecrosis. Promoter sequence variations can affect expression levels of PAI-1 gene and may disrupt the coagulation/fibrinolytic equilibrium, which may finally culminate into osteonecrosis. Hence, the aim of the study was to investigate the role of single-nucleotide polymorphisms (SNPs) in the promoter region of PAI-1 gene and osteonecrosis development. Methods: Two SNPs of the PAI-1 gene (rs2227631, -844 G/A; rs1799889, -675 4G/5G) were genotyped in 25 patients diagnosed with idiopathic ONFH and 25 control subjects, using direct sequencing. Subsequently, association analyses were performed for the genotyped SNPs. Results: Both the rs2227631 and rs1799889 genotype and allele frequencies of PAI-1 gene showed an insignificant association with osteonecrosis risk (P=0.717, 0.149). Haplotype frequencies of rs2227631 and rs1799889 were also calculated in patients having idiopathic ONFH and controls. Although the distribution of haplotype GA-4G 4G was found to be the highest among the cases, it was not significantly different when compared with the controls. Interpretation & conclusions: Our findings demonstrate that the minor alleles of promoter region sequences of the PAI-1 gene do not contribute to an increase in ONFH predisposition. However, this is a preliminary study and its findings should be considered as suggestive for studies to be done in a larger sample size

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish.

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration

Stereological investigation and expression of calcium-binding proteins in developing human inferior colliculus

The mammalian inferior colliculus (IC) is a major relay nucleus in the auditory pathway. Prenatal development of the human IC has been inadequately studied. The present study reports the morphometric development and maturation of the human IC using unbiased stereology, in 18 aborted fetuses of various gestational ages (12-29 weeks) and two babies aged 40 postnatal days (PND) and 5 months (that died of postoperative complications). It also demonstrates the functional maturation of the IC by examining the expression of calcium-binding proteins - parvalbumin (PV) and calbindin (CB). There was a significant increase in the total number of neurons and glia from 18 weeks of gestation (WG). The glia and neuron volume increased significantly from 16 WG to 22 WG, respectively. The total volume of IC also increased significantly from 18 WG onwards. On the other hand, the number and volume of undifferentiated cell bodies across all ages decreased significantly. Expression of CB was concentrated in the dorsal cortex while that of PV was mainly confined to the central nucleus of the IC, possibly indicating an early segregation of parallel processing of information in the auditory pathways. Intense staining for CB in the soma and dendrites appeared earlier than that of the PV. The morphological maturation appeared to overlap the onset of functional maturation suggesting an activity-dependent mechanism in the development of IC

Temporal distribution of mRNA expression levels of various genes in the developing human inferior colliculus

The inferior colliculus (IC) is a major binaural integration center in the auditory pathway. Interestingly, studies on the prenatal development of the human IC are lacking. During development of the nervous system a large repertoire of proteins is involved in transforming simple neuroblast cells into functional elements of the adult neural circuits. The present study reports on the mRNA levels produced by 12 genes involved in pre- [12-29 weeks of gestation (WG)], postnatal [40 postnatal days (PND) as well as 2 and 5 postnatal months (PNM)] developing human IC. The mRNA expression levels of nestin, vimentin, GFAP and DCX during 12-24 WG indicate the stages of neurogenesis, migration and differentiation of the human fetal IC. A decrease in the GAP-43 mRNA levels along with an increase in synaptophysin and PSD-95 mRNA levels during late gestational ages (24-29 WG) suggests the formation of primitive contacts by neurons with their targets and the onset of synapse formation. Expression levels of EGAD mRNA were transient with an increase in the early gestational ages, whereas that of GAD-67 mRNA increased in late gestational ages, indicating the changing role of GABA from a trophic factor to that of a neurotransmitter. High levels of BDNF, NT-3 and MBP mRNA in the late gestational ages reveal that the human IC undergoes neuronal maturation, synaptogenesis and myelination by 29 WG. Therefore, it may be suggested that the morphological maturation of the human IC occurs between 22 and 29 WG and that this period appears to be critical in the shaping of adult-like physiological attributes

Analysis of radial glial subtypes in uninjured and injured zebrafish spinal cord

<p><b>:</b> A-D) Transverse section of an uninjured cord showing GLAST⁺ (A, curved arrow), BLBP⁺ (B, arrow) radial glia counter stained with DAPI (C) and the merge picture (D). D.1) Higher magnification of boxed area of D showing radial glia colocalized with GLAST and BLBP. E-H) Transverse section of an uninjured cord showing GLAST⁺ (E, curved arrow), GFAP⁺ (F, arrow) radial glia stained with DAPI (G) and the merge image (H). H.1) Higher magnification of boxed area in section H showing a radial glia colocalized with GLAST and GFAP (curved arrow), and another radial glia stained with only GFAP (arrow). I-K) A section of 7 dpi cord showing GLAST⁺ radial glia (I, arrowheads), same GLAST⁺ radial glia colocalized with BrdU (J, small arrow) and its DIC image (K). L-N) A 7 dpi cord section showing BLBP⁺ radial glia (L, arrowheads), same BLBP⁺ radial glia colocalized with BrdU (M, small arrow) and its DIC image (N). O-Q) Higher magnification of a 7 dpi cord section showing GFAP⁺ radial glia with its process emerged from central canal (cc) towards pial membrane (O, arrowheads), same GFAP⁺ radial glia colocalized with BrdU (P, arrow) and its DIC image (Q). R) Drawing represents the time frame of SCI, BrdU treatment and tissue collection for cell proliferation experiments. S-U) Representative bar graph indicate quantification of GFAP⁺/BrdU⁺ cells (S), GLAST⁺/BrdU⁺ cells (T), BLBP⁺/BrdU⁺ cells (U) in uninjured, 3 dpi and 7 dpi cord. Values represent as mean ± s.e.m. (n = 5). Statistical significance represented as p value (Student’s t-test; **p<0.01, ***p<0.001). V and V1) Ultrastructure of a young glial progenitor cell with cytoplasmic processes containing intermediate filaments (white arrow, IF) and glycogen granules (white lines, GG) resembling radial glia in the ependymal region of a 7 dpi cord section. Inset V1 showing glycogen granules in the cytoplasm of a radial glia like cells also shown in V. W and W1) Ultrastructure of a 7 dpi cord section at the stump region shows aggregation of many ependymal cells (yellow arrowheads) with conspicuous microvilli (red arrow) protruding towards the central canal (cc). Inset W1 showing higher magnification of an ependymal cells with microvilli (MV, red arrow). X and X1) A 7 dpi cord section in the grey matter region showing many cytoplasmic processes of glia (green arrows) with compact intermediate filaments (IF) in the close vicinity of newly formed cells (red stars). Inset X1 indicating intermediate filaments in glial cytoplasm. In the insets, red boxed area inside the cartoon of injured spinal cord indicates the anatomical location of ultrastructural images. ‘NU’ and ‘CY’ denote nucleus and cytoplasm of the cell anatomy respectively. Scale bar = 50 μm (A-D, E-H); 20 μm (D.1, H.1); 15 μm (I-K, L-N); 5 μm (O-Q); 2 μm (V-X); 200 nm (V1-X1).</p