Effects of a High-Protein Diet on Kidney Injury under Conditions of Non-CKD or CKD in Mice.

Considering the prevalence of obesity and global aging, the consumption of a high-protein diet (HPD) may be advantageous. However, an HPD aggravates kidney dysfunction in patients with chronic kidney disease (CKD). Moreover, the effects of an HPD on kidney function in healthy individuals are controversial. In this study, we employed a remnant kidney mouse model as a CKD model and aimed to evaluate the effects of an HPD on kidney injury under conditions of non-CKD and CKD. Mice were divided into four groups: a sham surgery (sham) + normal diet (ND) group, a sham + HPD group, a 5/6 nephrectomy (Nx) + ND group and a 5/6 Nx + HPD group. Blood pressure, kidney function and kidney tissue injury were compared after 12 weeks of diet loading among the four groups. The 5/6 Nx groups displayed blood pressure elevation, kidney function decline, glomerular injury and tubular injury compared with the sham groups. Furthermore, an HPD exacerbated glomerular injury only in the 5/6 Nx group; however, an HPD did not cause kidney injury in the sham group. Clinical application of these results suggests that patients with CKD should follow a protein-restricted diet to prevent the exacerbation of kidney injury, while healthy individuals can maintain an HPD without worrying about the adverse effects.

Assessment of the subcutaneous degradation process of insoluble hyaluronic acid in rats.

Insoluble hyaluronic acid (IHA) may prevent adhesions by forming a physical barrier during the period when postoperative adhesions form. This study was performed to verify the changes that a solid IHA membrane undergoes as it is degraded in vivo, and to ascertain the swelling rate of IHA required for it to function as a physical barrier during the postoperative adhesion formation period. Nine female WI rats weighing 300-400 g were used. Discs 8 mm in diameter were cut out of dry IHA membranes made of IHA with a swelling rate (wet weight/dry weight) of either 2.47 (high-swelling IHA) or 1.94 (low-swelling IHA). They were placed in saline to swell and then washed with saline before subcutaneous implantation in four pockets in each rat. The high-swelling IHA started to degrade more rapidly than the low-swelling IHA. There was no evidence of degradation of the low-swelling IHA until day 7, but once it had started, the speed of degradation tended to be similar to that of the high-swelling IHA. The present results showed that, when IHA is implanted subcutaneously in rats, it is degraded over time in a phased process. The swelling rate required for the use of IHA as a postoperative adhesion barrier was also suggested.

Spinal cord stimulation modulates supraspinal centers of the descending antinociceptive system in rats with unilateral spinal nerve injury.

BACKGROUND: The descending antinociceptive system (DAS) is thought to play crucial roles in the antinociceptive effect of spinal cord stimulation (SCS), especially through its serotonergic pathway. The nucleus raphe magnus (NRM) in the rostral ventromedial medulla is a major source of serotonin [5-hydroxytryptamine (5-HT)] to the DAS, but the role of the dorsal raphe nucleus (DRN) in the ventral periaqueductal gray matter is still unclear. Moreover, the influence of the noradrenergic pathway is largely unknown. In this study, we evaluated the involvement of these serotonergic and noradrenergic pathways in SCS-induced antinociception by behavioral analysis of spinal nerve-ligated (SNL) rats. We also investigated immunohistochemical changes in the DRN and locus coeruleus (LC), regarded as the adrenergic center of the DAS, and expression changes of synthetic enzymes of 5-HT [tryptophan hydroxylase (TPH)] and norepinephrine [dopamine ß-hydroxylase (DßH)] in the spinal dorsal horn. RESULTS: Intrathecally administered methysergide, a 5-HT1- and 5-HT2-receptor antagonist, and idazoxan, an α2-adrenergic receptor antagonist, equally abolished the antinociceptive effect of SCS. The numbers of TPH-positive serotonergic and phosphorylated cyclic AMP response element binding protein (pCREB)-positive neurons and percentage of pCREB-positive serotonergic neurons in the DRN significantly increased after 3-h SCS. Further, the ipsilateral-to-contralateral immunoreactivity ratio of DßH increased in the LC of SNL rats and reached the level seen in naïve rats, even though the number of pCREB-positive neurons in the LC was unchanged by SNL and SCS. Moreover, 3-h SCS did not increase the expression levels of TPH and DßH in the spinal dorsal horn. CONCLUSIONS: The serotonergic and noradrenergic pathways of the DAS are involved in the antinociceptive effect of SCS, but activation of the DRN might primarily be responsible for this effect, and the LC may have a smaller contribution. SCS does not potentiate the synthetic enzymes of 5HT and norepinephrine in the neuropathic spinal cord.

Postnatal development of thalamic reticular nucleus projections to the anterior thalamic nuclei in rats.

The thalamic reticular nucleus (TRN) projects inhibitory signals to the thalamus, thereby controlling thalamocortical connections. Few studies have examined the development of TRN projections to the anterior thalamic nuclei with regard to axon course and the axon terminal distributions. In the present study, we used parvalbumin (PV) immunostaining to investigate inhibitory projections from the TRN to the thalamus in postnatal (P) 2- to 5-week-old rats (P14-35). The distribution of PV-positive (+) nerve fibers and nerve terminals markedly differed among the anterior thalamic nuclei at P14. Small, beaded nerve terminals were more distributed throughout the anterodorsal nucleus (AD) than in the anteroventral nucleus (AV) and anteromedial nucleus (AM). PV+ fibers traveling from the TRN to the AD were observed in the AV and AM. Nodular nerve terminals, spindle or en passant terminals, were identified on the axons passing through the AV and AM. At P21, axon bundles traveling without nodular terminals were observed, and nerve terminals were distributed throughout the AV and AM similar to the AD. At P28 and P35, the nerve terminals were evenly distributed throughout each nucleus. In addition, DiI tracer injections into the retrosplenial cortex revealed retrogradely-labeled projection neurons in the 3 nuclei at P14. At P14, the AD received abundant projections from the TRN and then projected to the retrosplenial cortex. The AV and AM seem to receive projections with distinct nodular nerve terminals from the TRN and project to the retrosplenial cortex. The projections from TRN to the AV and AM with nodular nerve terminals at P14 are probably developmental-period specific. In comparison, the TRN projections to the AD at P14 might be related to the development of spatial navigation as part of the head orientation system.

Chondroitinase ABC Administration Facilitates Serotonergic Innervation of Motoneurons in Rats With Complete Spinal Cord Transection.

Chondroitinase ABC (ChABC) is an enzyme that degrades glycosaminoglycan side-chains of chondroitin sulfate (CS-GAG) from the chondroitin sulfate proteoglycan (CSPG) core protein. Previous studies demonstrated that the administration of ChABC after spinal cord injury promotes nerve regeneration by removing CS-GAGs from the lesion site and promotes the plasticity of spinal neurons by removing CS-GAGs from the perineuronal nets (PNNs). These effects of ChABC might enhance the regeneration and sprouting of descending axons, leading to the recovery of motor function. Anatomical evidence, indicating that the regenerated axons innervate spinal motoneurons caudal to the lesion site, however, has been lacking. In the present study, we investigated whether descending axons pass through the lesion site and innervate the lumbar motoneurons after ChABC administration in rats with complete spinal cord transection (CST) at the thoracic level. At 3 weeks after CST, 5-hydroxytryptamine (5-HT) fibers were observed to enter the lesion in ChABC-treated rats, but not saline-treated rats. In addition, 92% of motoneurons in the ventral horn of the fifth lumbar segment (L5) in saline-treated rats, and 38% of those in ChABC-treated rats were surrounded by chondroitin sulfate-A (CS-A) positive structures. At 8 weeks after CST, many 5-HT fibers were observed in the ventral horn of the L5, where they terminated in the motoneurons in ChABC-treated rats, but not in saline-treated rats. In total, 54% of motoneurons in the L5 ventral horn in saline-treated rats and 39% of those in ChABC-treated rats were surrounded by CS-A-positive structures. ChABC-treated rats had a Basso, Beattie, and Bresnahan (BBB) motor score of 3.8 at 2 weeks, 7.1 at 3 weeks, and 10.3 at 8 weeks after CST. These observations suggest that ChABC administration to the lesion site immediately after CST may promote the regeneration of descending 5-HT axons through the lesion site and their termination on motoneurons at the level of caudal to the lesion site. ChABC administration might facilitate reinnervation by degrading CS-GAGs around motoneurons. Motor function of the lower limbs was significantly improved in ChABC-treated rats even before the 5-HT axons terminated on the motoneurons, suggesting that other mechanisms may also contribute to the motor function recovery.

Chondroitin sulfate expression around motoneurons changes after complete spinal transection of neonatal rats.

Hind limb locomotor activity spontaneously recovers after complete spinal transection (CST) in neonatal rats, but the mechanisms underlying the recovery are poorly understood. The perineuronal net (PNN) surrounding the neuronal cell bodies comprises an extracellular matrix that regulates neuronal plasticity during development. Here, we examined the expression of chondroitin sulfate (CS), a major component of the PNN, on motoneurons after CST in neonatal rats, and compared it with that in juvenile rats, in which hindlimb locomotor activity does not recover spontaneously. The spinal cord was transected at the mid-thoracic level in neonatal (postnatal day 5 [P5] and P10) and juvenile (P15 and P20) rats. Two weeks after CST, the percentage of motoneurons surrounded by chondroitin sulfate C (CS-C) - positive structures was significantly lower in rats with CST at P10 than in intact rats, and tended to be higher in rats with CST at P15 than in intact rats. The percentage of motoneurons with CS-A - positive structures was significantly lower in rats with CST at P15 than in intact rats. These findings suggest that CS-A and CS-C are differentially expressed in the PNNs in rats with CST. The decrease in CS-C - positive PNNs might facilitate the formation of new synaptic contacts to motoneurons, resulting in the recovery of the hindlimb locomotor activity in rats with CST during the neonatal period.

A retrograde tracing study of compensatory corticospinal projections in rats with neonatal hemidecortication.

To examine the compensatory mechanisms in rats that underwent left decortication at postnatal day 7 (P7), we injected the retrograde tracers fluorescein isothiocyanate-cholera toxin B subunit (FITC-CTB) and Fast Blue (FB) into the right and left upper cervical spinal cord, respectively, at postoperative weeks 2, 3, 4, and 5 and counted the number of retrogradely labeled corticospinal neurons in the right cerebral cortex compared with that in normally developed rats. Significantly more ipsilaterally projecting neurons were labeled with FITC-CTB in the decorticated rats compared with normal rats at all time points examined. The number of labeled neurons was similar to that at P7 in normal rats. There were also some FITC-CTB and FB double-labeled neurons in both decorticated and normal rats. The number of double-labeled neurons in the decorticated rats increased each week and was significantly greater than that in normal rats at postoperative weeks 4 and 5. The present results suggest that the elimination of ipsilaterally projecting axons observed in normal rats was prevented in the decorticated rats, so that the cerebral cortex neurons on the unlesioned side projected corticospinal tracts to the ipsilateral spinal cord. Furthermore, the collaterals of the corticospinal tracts originating from the cerebral cortex on the unlesioned side also project to the ipsilateral spinal cord. These compensatory mechanisms might underlie the acquisition of motor function in these animals.

Chondroitin sulfate expression around spinal motoneurons during postnatal development in rats.

Perineuronal nets are extracellular matrix structures that surround neuronal cell bodies and their proximal dendrites in the central nervous system. Chondroitin sulfate proteoglycans, which contain chondroitin sulfates (CSs) are major components of perineuronal nets. CSs are considered to have inhibitory roles in neural plasticity, although the effects differ according to their sulfation pattern. In the present study, we investigated the expression of the CS subtypes CS-A and CS-C surrounding spinal motoneurons in different postnatal periods to explore the potential influence of altered CS sulfation patterns on spinal development. CS-A-positive structures were observed around motoneurons in the cervical, thoracic, and lumbar segments as early as postnatal day (P) 5. Most motoneurons were covered with CS-A-positive structures during the first 2 postnatal weeks. The percentage of motoneurons covered with CS-A-positive structures decreased after P20, becoming lower than 70% in the cervical, and lumber segments after P35. CS-C-positive structures were occasionally observed around motoneurons during the first 2 postnatal weeks. The percentage of motoneurons covered with CS-C-positive structures increased after P20, becoming significantly higher after P25 than before P20. The expression pattern of Wisteria Floribunda agglutinin-positive structures around motoneurons was similar to that of the CS-C-positive structures. The present findings revealed that CS-A and CS-C are differentially expressed in the extracellular matrix surrounding motoneurons. The altered sulfation pattern with increased CS-C expression is associated with the maturation of perineuronal nets and might lead to changes in the motoneuron plasticity.

Neonatal spinal injury induces de novo projections of primary afferents to the lumbosacral intermediolateral nucleus in rats.

Complete spinal transection in adult rats results in poor recovery of hind limb function and severe urinary bladder dysfunction. Neonatal rats with spinal cord transection, however, exhibit spontaneous and significant recovery of micturition control. A previous study in which biotinylated-dextran amine (BDA) was used as an anterograde tracer demonstrated that primary afferent fibers from the fifth lumbar dorsal root ganglion (DRG) project more strongly and make more terminals in the ventral horn after neonatal spinal cord transection at the mid-thoracic level. In the present study, we injected BDA into the sixth lumbar (L6) DRG of neonatally spinalized rats to label primary afferent fibers that include visceral afferents. The labeled fibers projected to the intermediolateral nucleus (IML) in the intermediate zone on ipsilateral side of the L6 spinal segment, whereas no projections to the IML were observed in sham-operated or intact rats. The BDA-labeled fibers of neonatally spinalized rats formed varicose terminals on parasympathetic preganglionic neurons in the IML. These findings suggest that some primary afferent projections from the L6 DRG to the IML appear after neonatal spinal cord transection, and these de novo projections might contribute to the recovery of autonomic function such as micturition following spinal cord injury in the neonatal stage.

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain.

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.