Age-related changes in calretinin-immunoreactive periglomerular cells in the rat main olfactory bulb

The changes of calretinin (CR)-immunoreactive periglomerular cells in the glomerular layer of the main olfactory bulb (MOB) were investigated in rats differing ages from postnatal month 1 (PM 1) to PM 24. The number of cresyl violet-positive periglomerular cells was similar between PM 1 and PM 12, but they decreased slightly in the PM 24 group. The size of CR-immunoreactive periglomerular cells in the glomerular layer increased with age, while their numbers did not change significantly in the PM 6-PM 24 groups. In the PM 24 group, numbers of CR-positive periglomerular cell bodies and their processes decreased, while the size of CR-positive cell bodies in the glomeruli was larger than that of the previous groups. These results suggest that CR-immunoreactive periglomerular cells in the rat MOB are well-developed in the PM 6 group, and that periglomerular cells in the PM 24 group show poor CR-immunoreactivity compared to those in the PM 6 group.     

Immunohistochemical localization of glutamate in the gerbil main olfactory bulb using an antiserum directed against glutamate

Information on the localization and the roles of glutamate in the nervous system is becoming valuable because the axon terminals of the olfactory sensory neurons and the synapses of the mitral and tufted output cells appear to be glutamatergic. In this study, we have analysed the distribution of glutamate immunoreactivity in the main olfactory bulb (MOB) of the Mongolian gerbil using an antiserum directed against glutamate. Glutamate immunoreactivity in the MOB was present in the olfactory nerve layer (Onl), glomerular layer (GL), external plexiform layer (EPL) and mitral cell layer (ML), but not in the granule cell layer (GCL). Glutamate immunoreactivity detected in the Onl was thought to be terminal ramifications of glomeruli. Some neurons in the periglomerular region showed glutamate immunoreactivity. In the EPL, glutamate immunoreactivity was found in some neuronal somata (tufted cells) and processes. In addition, mitral cells in the ML were labelled by the glutamate antibody. The pattern of glutamate immunoreactivity in the mitral cells was similar to that in the tufted cells. In brief, glutamate in the gerbil MOB is the neurotransmitter used by primary afferents and output neurons.     

Histochemical study on neurodegeneration in the olfactory bulb after transient forebrain ischaemia in the Mongolian gerbil

In the present study, we investigated the ischaemia-related neurodegeneration in the main and accessory olfactory bulb (AOB) after 5 min transient forebrain ischaemia in the Mongolian gerbil using the acid fuchsin staining method. Between 5 and 15 days after ischaemia, acid fuchsin positive cells markedly increased in the external plexiform layer (EPL), mitral cell layer (ML) and glomerular layer (GL) of the main olfactory bulb (MOB), and in the mixed cell layer (MCL) and GL of the AOB. By 30 days after ischaemia reperfusion, acid fuchsin positive neurons were shrunken and showed low acidophilia in somata. Many necrotic vacuoles were found in the EPL and GL of the MOB 30 days after ischaemia. At this time, necrotic vacuoles were very few in the AOB. Therefore, our results suggest that the GL and EPL of the MOB are vulnerable to ischaemic damage at a later time after ischaemic insult, and that the AOB is more resistant to ischaemic damage as compared with the MOB.     

Morphological characteristics of C1 and C2 adrenergic neurone groups in marmoset monkey brainstem by using antibody against phenylethanolamine-N-methyltransferase

This work describes a mapping study of phenylethanolamine-N-methyltransferase (PNMT) immunoreactive neurones and fibres in the medulla oblongata of the marmoset monkey, Callithrix jacchus. Two groups of PNMT-immunoreactive neurones were found in the marmoset monkey medulla oblongata: a ventrolateral (C1 group) and a dorsomedial PNMT-immunoreactive cells group (C2 group). The PNMT-immunoreactive cells in the ventrolateral group C1 were found to be located around the lateral reticular nucleus. The PNMT-immunoreactive somata within the ventrolateral medulla are round to oval, and mostly multipolar with branched processes. In the dorsomedial group C2, PNMT-immunoreactive cell bodies appeared near the obex. The majority of the dorsomedial PNMT-immunoreactive neurones were observed in the nucleus tractus solitarius; although some were present in the dorsal motor nucleus of the vagus. The PNMT-immunoreactive somata in the dorsomedial medulla were small and round or ovoid. These results provide information upon the adrenergic system in the medulla oblongata of a species that presents a useful model of a small primate brain, the marmoset monkey.     

Projections from the anterior interposed nucleus to the red nucleus diminish with age in the mouse

To analyse the effect of ageing on the projection of the anterior interposed nucleus to the red nucleus, we injected the retrograde tracer fluorogold in the red nucleus of 3-, 6- and 12-month-old mice. The number of labelled neurones in the anterior interposed nucleus fell by 9% between 3 and 6 months and by another 9% between 6 and 12 months (all P < 0.001). This suggests that loss of neurones from the cerebellar nuclei starts well before old age.     

Post-natal changes of cyclin-dependent kinase 5 activator expression in the developing rat cerebellum

cDNA of cyclin-dependent kinase 5 (Cdk5) was cloned based on its primary sequence homology to Cdc2 and Cdk2. Cdk5 requires the neuronal Cdk5 activators such as p35 or p39(nck5ai) (p39) for its activity. In this study, we examined post-natal changes in the p39 expression pattern during the development of the rat cerebellum. p39 began to express in somata and dendrites of Purkinje cells at post-natal day 3 (PD3). In particular, at PD12, parasagittal bands (stripes) with p39 immunoreactivity were weakly observed. At PD21, p39-immunoreactive stripes were developed when compared with the PD12 group. At this age stage, p39 immunoreactivity became weak in somata of Purkinje cells, not forming stripes. At PD28, a series of parasagittal bands were more distinct than those of the PD21 group, and p39 immunoreactivity disappeared in Purkinje cells, not forming p39 immunoreactive stripes. In the adults, p39 immunoreactivity in Purkinje cells was similar to that found in the PD28 group which showed that parasagittal bands were very narrow, and became progressively more slender. Therefore, we suggest that the post-natal changes of p39 expression in Purkinje cells in the cerebellum is an autonomous characteristic of Purkinje cells with a role of Cdk5 activators.     

Quantitative analysis of cells in the ganglion cell layer of the chick retina: developmental changes in cell density and cell size

Changes in cell density and size in the ganglion cell layer (GCL) of the retina were studied in chick embryos and post-hatching chicks. The total number of cells in the GCL increased from 3.64 million at embryonic day 8 (E8) to the maximal 7.85 million at E14. After E14, the number of cells decreased to 6.08 million at post-hatching day 1 (P1) and 4.87 million at P8. Cell density in the GCL decreased unevenly according to retinal regions; cell density in the presumptive central area (pCA) of P8-chicks decreased to approximately 45% of that in E8-embryos. Densities of the nasal peripheral retina (NP) and temporal peripheral retina (TP) of P8-chicks decreased to 23 and 18% of E8-embryos, respectively. Differentiation of the central (44,000 cells/mm(2) in pCA) - peripheral (28,000 cells/mm(2) in TP) gradient in cell density was formed by E8. The presumptive dorsal area (pDA) was shaped by E11, but became obscure with age. Although ganglion cell sizes were basically uniform at E8, differentiation occurred with the appearance of larger ganglion cells after E14. Mean size of retinal ganglion cells increased 2.8-fold in the pCA and 3.8-fold in the TP between E8 and P8, accompanying a similar scale of decreases in cell densities.     

Pituitary adenylate cyclase-activating polypeptide-immunoreactive cells in the ageing gerbil hippocampus

In the present study, we investigated age-related changes in pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity and its protein levels in the gerbil hippocampus at various ages using immunohistochemistry and western blot analysis. In the post-natal month 1 (PM 1) group, PACAP-immunoreactive cells were found in all hippocampal subregions. The number of PACAP-immunoreactive cells was decreased in the PM 3 group and was still more decreased in the PM 6 and 12 groups. Thereafter, in the PM 18 and 24 groups, PACAP-immunoreactive cells were significantly increased again. However, in the mossy fibre zone, PACAP immunostaining was very strong in the adult group, especially in the PM 6 group. In addition, PACAP protein level was highest at PM 6, showing a slight decrease at PM 24. These results indicate that PACAP-immunoreactive cells are lowest in the adult stage and highest in the aged stage. However, PACAP immunoreactivity in the mossy fibre zone and PACAP protein level in the hippocampus are highest in the adult stage.     

Distribution of serotonin immunoreactivity in the main olfactory bulb of the Mongolian gerbil

The distribution of serotonin immunoreactivity in the main olfactory bulb (MOB) of the Mongolian gerbil (Meriones unguiculatus) was examined by immunohistochemistry. Seven distinct layers of the Mongolian gerbil MOB-stained with cresyl violet were identified. Serotonin-immunoreactive (IR) cell bodies were not found in the MOB. The serotonin-IR nerve fibres had a specific laminar distribution and morphology in the gerbil MOB. Serotonin-IR nerve fibres were observed in the glomerular, external plexiform and granule cell layers of the MOB. These serotonin-IR nerve fibres showed varicosities that were larger than the thickness of the axon. The highest density of serotonin-IR nerve fibres was in glomeruli of the glomerular layer. The average fibre density in the glomerular layer was more than three to four times the density in the infraglomerular layers. Glomerular serotonin-IR fibres were much more intensively stained than infraglomerular serotonin-IR fibres. This result suggests that serotonin-IR nerve fibres of Mongolian gerbil MOB are extrinsic and may act to modulate the olfactory transmission.     

Histochemical and electron microscopic study on motor neurone degeneration following transient spinal cord ischaemia at normothermic conditions in rabbits

This study was carried out to investigate the motor neurone degeneration in the ventral horn following transient spinal cord ischaemia at normothermic conditions in rabbits. Transient spinal cord ischaemia was induced by occlusion of the abdominal aorta underneath the left renal artery for 15 min at normothermia (38.7 degrees C). Sections at the level of L7 were examined using histochemical and electron microscopic methods. Cresyl violet-positive motor neurones began to reduce in number at 3 h after ischaemia reperfusion, and were not detectable at 48 h after ischaemia reperfusion. Acid fuchsin-positive motor neurones were detected at 1 h after ischaemia reperfusion, significantly increased up to 6 h after the ischaemia reperfusion, and eventually disappeared by 48 h after ischaemia reperfusion. In electron microscopic findings, the disintegration of cytoplasmic membranes, and the disruption of mitochondria and endoplasmic reticulum were observed in motor neurones at 30 min after ischaemia reperfusion. Motor neurones showed necrotic findings with pyknotic degeneration at 1 h after ischaemia reperfusion. The necrotic degeneration became severer time dependently after ischaemia reperfusion. At 48 h after ischaemia reperfusion, cellular components were not detectable in motor neurones. In conclusion, we suggest that the degeneration pattern of motor neurones of the ischaemic spinal cord was necrotic after ischaemia reperfusion under normothermic conditions.     

Phylogenic aspects of the amphibian dual olfactory system

The phylogenic significance of the subdivision of dual olfactory system is reviewed mainly on the basis of our findings by electron microscopy and lectin histochemistry in the three amphibian species. The dual olfactory system is present in common in these species and consists of the projection from the olfactory epithelium (OE) to the main olfactory bulb (MOB) and that from the vomeronasal epithelium (VNE) to the accessory olfactory bulb (AOB). The phylogenic significance of subdivisions in the dual olfactory system in the amphibian must differently be interpreted. The subdivision of the MOB into its dorsal region (D-MOB) and ventral region (V-MOB) in Xenopus laevis must be attributed to the primitive features in their olfactory receptors. The middle cavity epithelium lining the middle cavity of this frog possesses both ciliated sensory cells and microvillous sensory cells, reminding the OE in fish. The subdivision of the AOB into the rostral (R-AOB) and caudal part (C-AOB) in Bufo japonicus formosus must be regarded as an advanced characteristic. The lack of subdivisions in both MOB and AOB in Cynops pyrrhogaster may reflect their phylogenic primitiveness. Since our lectin histochemistry to detect glycoconjugates expressed in the olfactory pathway reveals the subdivisions in the dual olfactory system in the amphibian, the glycoconjugates may deeply participate in the organization and function of olfactory pathways in phylogeny.     

Vascularization of the Ciliary Body and Iridocorneal Angle in the Avian Eye

Most mammals have two different structures in which we found glomerular layers at the same time: the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB). Both bulbs have the same pattern of organization, but there are some differences: although the size is considerably bigger in MOB than in AOB, probably the most important difference is that the principal cells are not differentiated into mitral and tufted cells in the AOB, and are usually described as mitral/tufted cells. We have previously observed that in some mammals, like pigs and sheep, the AOB reaches maturity before birth, but this is not a rule for other species. Surprisingly, mice need several days of life to achieve full stratification of its cellular components. We have studied the chronology of this process, focusing our attention on the glomerular layer, the last to appear. We concluded that there are two critical periods, between E11.5 and E16.5 (migration phase) and between E17.5 and P3.5–7 (true morphological constitution).     

Morphogenesis of the olfactory pit in a flatfish, barfin flounder (Verasper moseri)

Morphogenesis of the olfactory pit (OP), olfactory lamella (OL) and olfactory epithelium (OE) was examined by scanning electron and light microscopy in the barfin flounder (Verasper moseri). At day 0 after hatch, the OP was already formed. At day 14, the cellular differentiation of the OE was prominent. At day 42, the OP became a cavity by the formation of its roof. At day 56, the first OL extended remarkably and was lined with the OE on both sides. The OL increased in number with development. These findings suggest that the OE is functionally active at day 14. The formation of the OL in the OP may be initiated by the stimulus when the barfin flounder touched at the bottom of the sea.     

Secondary or Accessory Glomerular Layer in Mice During Growth

Most mammals have two different structures in which we found glomerular layers at the same time: the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB). Both bulbs have the same pattern of organization, but there are some differences: although the size is considerably bigger in MOB than in AOB, probably the most important difference is that the principal cells are not differentiated into mitral and tufted cells in the AOB, and are usually described as mitral/tufted cells. We have previously observed that in some mammals, like pigs and sheep, the AOB reaches maturity before birth, but this is not a rule for other species. Surprisingly, mice need several days of life to achieve full stratification of its cellular components. We have studied the chronology of this process, focusing our attention on the glomerular layer, the last to appear. We concluded that there are two critical periods, between E11.5 and E16.5 (migration phase) and between E17.5 and P3.5–7 (true morphological constitution).     

Regional specialization of ganglion cell layer of the chick retina

Specialization of the ganglion cell layer (GCL) was studied by Nissl-staining and axonal tract-tracing methods in chicks and chick embryos. The changes in the retinal area and the cell number in the GCL produced a disparity in the cell density that occurred through the two different processes, cell generation (before embryonic days 10-14, E10-14) and cell loss (after E10-14). One high-density area was found in the retinal fundus on E8 (presumptive central area, pCA) and its density decreased toward the peripheral retina. Another high-density area was found in the dorsal retina on E11 (presumptive dorsal area, pDA). Cell densities of the pCA and the pDA on E11 decreased gradually to 25-30% by P1, and after that they further decreased to 40-60% by P30. The pCA was still identified on P30, but the pDA became very obscure by this age. In contrast, ganglion cell sizes increased 5-7 times in the pCA and pDA from E8 to P30, and increased 12 times in the temporal periphery. The present study suggests that the center-peripheral gradient of cell density results from lager scale of cell genesis in the pCA, but not from lager scale of cell loss in the peripheral retina. However, obscuration of the pDA results from equalization of cell density in cellular degeneration processes.     

蛋白质组学技术解析急性免疫应激影响肉仔鸡肉品质的机理

旨在探讨免疫应激对肉仔鸡胸肌肉品质的影响机理。采用非标定量蛋白质组学技术研究免疫应激对AA肉仔鸡胸肌肉品质和蛋白质组学变化的影响。本试验选取120只1日龄健康AA肉仔公鸡，随机分为2个处理，分别为对照组和免疫应激处理组，每个处理6个重复，每个重复10只鸡。分别在36、38、40日龄时，于腹腔注射1 mL生理盐水（对照组）或5.0 mg·kg^-1体重的LPS溶液（免疫应激组）。42日龄时，每重复随机抽取2只鸡，采集胸肌肌肉组织测定滴水损失、蒸煮损失、肌苷酸含量和pH，及蛋白质组的定性、定量分析。结果显示：1）与对照组相比，免疫应激显著增加了肉鸡胸肌的滴水损失和蒸煮损失（P<0.05），显著降低了胸肌内肌苷酸（IMP）含量（P<0.05）；免疫应激对肉鸡胸肌宰后静置24和48 h的pH影响不显著（P>0.05）；免疫应激组肉鸡胸肌肌纤维的横截面周长和面积均极显著增加（P<0.01）。2）定性蛋白质组学分析结果显示，对照组特异表达蛋白质功能富集到了细胞呼吸、有氧呼吸、己糖代谢与生物合成过程、单糖生物合成过程、葡萄糖代谢过程和糖异生生物学过程；而免疫应激组特异表达蛋白质富集到了紧密连接代谢通路和大分子复合物分解、蛋白质分解与解聚、核酸合成和代谢、糖基化合物合成与代谢和含碱基小分子物质代谢生物学过程。3）定量蛋白质组学分析表明，免疫应激组和对照组有21个蛋白质发生差异化表达，其中15个蛋白质在免疫应激组上调表达，6个蛋白质在免疫应激组下调表达；免疫应激组上调表达的蛋白包括参与碳水化合物代谢和能量产生相关蛋白（AMPD1、GPD1L2、LDHA、UQCRC2）、肌肉收缩相关蛋白（MYH1C、MYH1A、CASQ2、TMEM38A）和参与免疫反应保护细胞免受损伤相关蛋白（HMGB1、PPP1R1A、LOC101747971、PRDX1、ABCF2）；免疫应激组下调表达的蛋白有HBBA、CS、ATP5A1WL、HSP90B1、ABCB6和SYNJ2。综上所述，LPS诱导的免疫应激通过改变肉鸡肌肉与能量代谢和肌肉收缩相关蛋白质的表达，从而增加了肌纤维的面积，并降低了肌细胞持水力，导致肉品质下降。     

Effect of single- and multiple-dose 0.5% timolol maleate on intraocular pressure and pupil size in female horses

OBJECTIVE: To determine the effect of single and multiple-dose 0.5% timolol maleate on intraocular pressure (IOP) and pupil size between 8 AM and 8 PM. Animals Nine female horses with normotensive eyes. Procedure IOP, horizontal and vertical pupil size were measured on a single day, between 8 AM and 8 PM at hours 0, 0.5, 1, 2, 4, 6, 8, 10, and 12. A single dose of 0.5% timolol maleate was applied to both eyes immediately after the first measurement at 8 AM. IOP and pupil size were measured at 8 AM and 4 PM in a 5-day experiment of twice-daily application of 0.5% timolol maleate. RESULTS: A significant decrease in IOP from 24.9 +/- 4.2 mmHg prior to application of timolol maleate to 20.7 +/- 3.1 mmHg (4.2 mmHg = 17%) was observed 8 h after single-dose application. A significant decrease in horizontal pupil size (2.0 mm = 11%) was present 6 h after single-dose application. In the multiple-dose experiment, a significant decrease in IOP was present on days 4 and 5 as compared to IOP measured prior to application of timolol maleate. A significant decrease in horizontal and vertical pupil size was present throughout the 5-day study as compared to the values obtained prior to treatment. CONCLUSIONS: 0.5% timolol maleate significantly decreased IOP and pupil size in normo-tensive eyes of this group of female horses in both single and multiple twice daily applications.     

Chronological alterations of P2X3 receptor expression in the trigeminal ganglion after ischaemic insult in the Mongolian gerbil

P2X receptors play a role in the transduction of sensory signals like pain. Few studies have been undertaken on altered P2X(3) receptor (P2X3) expression in sensory neurones after peripheral nerve injury. In the present study, we investigated chronological alterations in P2X3 immunoreactivity and its protein content in the trigeminal ganglion after ischaemic insult in the Mongolian gerbil. In the sham-operated group, P2X3-immunoreactive neurones were found abundantly in small- and medium-sized neurones. From 1 day after ischaemic insult, the number of P2X3-immunoreactive neurones decreased significantly. At 5 days after ischaemic insult, P2X3 immunoreactivity was observed in few neurones, but its immunoreactivity was weak. However, the number of cresyl violet-positive neurones was unchanged throughout this period in all groups. These results suggest that transient trigeminal ganglion ischaemia may provoke a decrease of P2X3 expression and its protein content, and that this down-regulation of P2X3 may be related to the altered pain and thermal sensation without being associated with a transient ischaemic insult.     

Tenderization potential of Hanwoo beef muscles from carcasses with differed genders and loin intramuscular fat content levels during post mortem ageing

Carcasses from Hanwoo steers (n = 15) and cows (n = 15) were classified into three groups: group 1 (G1), the carcasses had 10% to < 11.5% intramuscular fat (IMF) in loin muscles; group 2 (G2), the carcasses had 13% to < 14.5% IMF in loin muscles; and group 3(G3), the carcasses had 17% to < 18.5% IMF in loin muscles. These were used to evaluate the effects of gender and carcass group on quality traits and Warner–Bratzler shear force (WBSF) of Psoas major (PM), Longissimus thoracis (LT), Longissimus lumborum (LL), Longus colli (LC), Supraspinatus (SS), Latissimus dorsi (LAD), Semimembranosus (SM), Quadriceps femoris (QF), Biceps femoris (BF) and Semitendinosus (ST) muscles. Our results showed that pH values of LT, LL, LC, BF and QF muscles were lower in steers than in cows (P < 0.05). Water holding capacity (WHC) was found higher in LC, SS, LAD and QF muscles of steers (P < 0.05). At day 2 of ageing, gender affected the WBSF values of only PM, LD and QF muscles in G1, and QF muscle in G3; however, with additional ageing, the gender effect was observed for most of the muscles. Most muscles showed ageing responses; however, the rates of ageing response significantly varied depending on gender and carcass groups. The muscles of G1 and G2 had generally higher tenderization potentials than those of G3. Furthermore, most muscles in G3 had generally lower WBSF values than in G1 and G2. These results clearly indicate that ageing has a significant effect on quality and WBSF of beef muscles, and the classification by loin IMF level may be useful for prediction of the tenderness of other muscles.     

Age-related changes in rat optic nerve: morphological studies

Age-related changes of the optic nerve were studied in 3-month-old (young), 12-month-old (adult) and 24-month-old (aged) male Sprague-Dawley rats. Cross sections of the intracranial portion of the optic nerves of animals of different age groups were stained with haematoxylin-eosin and examined under a light microscope at low and high magnification. Other sections were stained with crystal violet for demonstration of glial cells. A third group of sections were stained immunohistochemically to detect glial fibrillary acidic protein (GFAP) which is a marker for localizing and characterizing astrocytes. All morphological results were subjected to the quantitative analysis of images and to statistical analysis to identify significant morphometrical data. Tissue protein concentrations were determined on homogenized fragments of optic nerve. Our results demonstrate the following age-related changes: (1) increase of the optic nerve sheaths (meningeal membranes); (2) increased number of astrocytes; (3) increase of areal density of GFAP immunoreactivity; (4) increased diameter and area of the optic nerve; (5) decreased number of nerve fibres; (6) decreased-size of nerve fibres and (7) decrease of the nerve fibres/meningeal membrane ratio from 3:1 to 1:1. Moreover, the protein amount does not change with age. The rat optic nerve, therefore, appears sensitive to ageing processes and can be considered as a useful model for the studies on neuronal ageing.