GPR17 plays a role in ischemia-induced endogenous repair of immature neonatal cerebral White matter.

Whether GPR17 has the same distribution and repair mechanism in immature white matter with periventricular leukomalacia (PVL) as in the adult brain remains to be determined. This study tried to explore the expression phase and site of GPR17, and to investigate the effect of silencing GPR17 on endogenous repair mechanism of immature white matter with PVL. Ischemic PVL in vivo results showed that GPR17 gene and protein expression increased more in the PVL than in the sham group at 12 h-24 h and 72h to 7 days after PVL. NG2+/GPR17+progenitor cells at 48 h-96 h and O4+/GPR17+precursor cells at 72h to 7d were also significantly increased in the PVL compared to the sham groups. Results in vitro showed that oxygen-glucose deprivation (OGD) also induced more GPR17 gene and protein expression than control at 48 h-72 h. There were more NG2+/GPR17+progenitor cells at 24 h-48 h and O4+/GPR17+precursor cells at 48 h-72 h in the OGD groups, as well. The functional role of GPR17 in the intrinsic repair response to ischemia was tested using GPR17 gene silencing. The progenitor cells and OL precursors in the OGD+GPR17 silencing group were both significantly less than those in the control, OGD and OGD+gene silencing control groups. The apoptotic percentage of cells in OGD+GPR17 silencing group was also much higher. In summary, ischemia-induced GPR17 expression was shown to contribute to glial-derived progenitor cell proliferation and differentiation into OL precursors, which may provide a therapeutic target for immature neonatal white matter injury after ischemia.

CaSR is required for ischemia-induced proliferation and differentiation of white matter progenitor cells from neonatal rats.

This study was designed to investigate whether calcium-sensing receptor (CaSR) could induce immture white matter progenitor cells proliferation and differentiation into oligodendrocyte(OL) precursor cells after oxygen-glucose deprivation (OGD) in vitro. Progenitor cells of immature white matter originating from five-day-old newborn rats were divided into control, OGD, control + CaSR silencing, OGD + CaSR silencing, control + adenosine triphosphate magnesium chloride (ATP-MgCl2) and OGD + ATP-MgCl2 groups. Immunofluorescence, real-time RT-PCR, gene silencing, Hoechst 33342/propidium iodide (PI) and Flow cytometry tests were used to examine the proliferation, differentiation and survival of the white matter progenitor cells in the different treatment groups. The results showed that normal immature white matter progenitor cells have certain ability of self-proliferation and differentiation in vitro. OGD could further induce progenitor cells proliferation and differentiation into O4 + OL precursor cells by activating CaSR, but OGD also induced more necrosis and apoptosis of newborn cells and less MBP + OL formation. The addition of ATP-MgCl2 as an activating agent of CaSR further promoted cell proliferation and differentiation both under normal and OGD conditions and reduced OGD-induced apoptosis and necrosis, while CaSR silenced resulted in minimal cell proliferation, differentiation and survival. This study suggests that CaSR plays an important role in the induction of immature white matter progenitor cells proliferation and differentiation into OL precursor cells after OGD, which may provide a new angle to further study whether CaSR initiates the intrinsic repair potential of immature white matter after ischemia in vivo.

White matter and SVZ serve as endogenous sources of glial progenitor cells for self-repair in neonatal rats with ischemic PVL.

Mounting evidence suggests that endogenous progenitor cells may initiate cerebral WM repair. This study was designed to determine whether endogenous glial progenitor cells derived from either the subventricular zone (SVZ) or the white matter (WM) contribute to WM repair in a neonatal rat model of ischemic periventricular leukomalacia (PVL). Additionally, the role of G protein-coupled receptor 17 (GPR17), recently shown to act as a sensor for WM damage, was explored to assess its potential recruitment and activation of endogenous glial progenitor cells for such WM self-repair. Our in vivo and in vitro models consisted of five-day-old neonatal rats or cultured glial progenitor cells derived from both the SVZ and WM of these rats, randomly divided into sham/control and induced ischemic PVL/oxygen-glucose deprivation (OGD) groups. The WM of all PVL rats showed either mild or severe histopathological changes, with significantly increased in vivo apoptosis and poor myelination compared to those of the sham group. Significantly more apoptotic and necrotic cells were also detected in the OGD glial progenitor cell cultures derived from the SVZ and WM at all time intervals. The glial progenitor cells were significantly increased in both the SVZ (NG2⁺/GPR17⁻/BrdU⁺) and WM (NG2⁺/GPR17⁺/BrdU⁺) within 72 h after PVL; preOLs were also increased significantly in both the SVZ (O4⁺/GPR17⁻/BrdU⁺) and WM (O4⁺/GPR17⁺/BrdU⁺) within 7d after PVL in vivo or OGD in vitro. However, the more differentiated CNPase⁺/GPR17⁻/BrdU⁺ and MBP⁺/GPR17⁻/BrdU⁺ OLs in the SVZ and WM remained significantly less than those in the sham groups up to 14d or 21d after OGD or PVL, respectively. Hence, both the WM and SVZ were found to be potential endogenous sources of glial progenitor cells for WM repair in PVL rats. However their endogenous self-repair capacity appeared to be limited, since the more mature OLs did not completely recover from experimental ischemia, even after 14-21d.

[Effect of single or combined application of UDP-glucose, GDNF and memantine on improvement of white matter injury in neonatal rats assessed with light and electron microscopy pathologically].

OBJECTIVE: To evaluate pathologically the effect of the single or combined application of UDP-glucose, GDNF and memantine on the improvement of white matter injury in neonatal rats with periventricular leukomalacia (PVL) under light and electron microscopy. METHODS: A five-day-old neonatal rat model for PVL was established by ligation of the lateral common carotid artery following 120-minute hypoxia. Rats were randomly divided into six groups (30 rats in each group): sham-operated, PVL, UDP-glucose (UDP-glucose 2000 mg/kg intraperitoneally after PVL), GDNF (GDNF 100 µg/kg intracerebrally after PVL), tmemantine (memantine 20 mg/kg intraperitoneally after PVL), and a combination administration of three drugs (UDP-glucose, GDNF and memantine). The rats were sacrificed 7 or 21 days after PVL for assessment of pathological changes in the white matter under both light and electron microscopy. The number and thickness of the myelin sheath in the white matter were measured under electron microscopy, and both of pathological grading and scoring were undertaken under light microscopy. RESULTS: There was rare and sparse myelinogenesis with a loose arrangement of nerve fibers in the white matter under electron microscopy in the PVL group at 7 and 21 days after PVL. The number and thickness of the myelin sheath in the PVL group were significantly less than in the sham-operated, UDP-glucose, GDNF, memantine and combination administration groups (P<0.01). The results of pathological grading of white matter under light microscopy showed that all rats in the PVL group manifested either mild injury (38%-50%) or severe injury (50%-62%) at 7 and 21 days after PVL. The majority of rats (50%-88%) in the four drug administration groups had normal white matter at 7 and 21 days after PVL. The pathological scores at 7 and 21 days after PVL in the PVL group were the highest, and they were significantly higher than in the other five groups (P<0.05). CONCLUSIONS: The single or combined application of UDP-glucose, GDNF and memantine may significantly improve pathological changes in the white matter of rats with PVL. The favorable effect is inferred to be closely correlated with the improvement of brain microenvironment and the enhancement of nerve regeneration promoted by the three drugs.

Periventricular leukomalacia long-term prognosis may be improved by treatment with UDP-glucose, GDNF, and memantine in neonatal rats.

The therapeutic effects of UDP-glucose (UDPG), an endogenous agonist of GPR17 that may promote the self-repair of white matter, glial cell line-derived neurotrophic factor (GDNF), a neurotrophic factor correlated with the growth and survival of nerve cells, and memantine, an antagonist of NMDA receptors, were evaluated for functional improvement of neonatal rats with experimental periventricular leukomalacia (PVL). Five day-old neonatal rat pups were subjected to an ischemia-induced model of PVL. The pups were then randomly divided into sham, PVL, PVL plus UDPG, PVL plus GDNF, and PVL plus memantine groups. All pups were weighed and the age at first eye opening recorded. Pathological changes and myelin sheath formation in the white matter were assessed under both light and electron microscopy on day 7 and 21 after induction of PVL. Values of escape latency (EL) and swimming distance (SD) in Morris water maze test, and the modified inclined plane scores in Rivlin inclined plane test were recorded for rats on day 26. Pups in the PVL group were found to be significantly lower in weight (p<0.05), delayed in age at first eye opening (p<0.01), and impaired in their inclined plane (p<0.01) and Morris water maze (p<0.01) performance compared with those in the sham, UDPG, GDNF and memantine groups. Histopathological grading of the white matter classified all pups in the PVL group with significantly more severe injury (p<0.01), and the number and thickness of their myelin sheaths were significantly less (p<0.01), compared to the UDPG, GDNF, memantine, or sham groups. These results indicate that treatment with UDPG, GDNF, and memantine may significantly improve long-term prognosis in neonatal rats with cerebral white matter injury, characteristic of PVL.

[Endogenous self-repair in immature white matter induced by ischemia in neonatal rats].

OBJECTIVE: To study in vivo the endogenous self-repair mechanism in immature white matter induced by ischemia in neonatal rats with periventricular leukomalacia (PVL). METHODS: Five-day-old neonatal Sprague-Dawley (SD) rats were randomly divided into sham and PVL groups. Rat model of PVL was prepared by ligation of the right common carotid artery following 2 hours of exposure to 8% oxygen. Pathological changes and myelination in the white matter were assessed under light and electron microscopy at 7 and 21 days after PVL. O4-positive OL precursor cells in the white matter were determined with immunofluorescence staining. Activation, proliferation, migration and differentiation of glial progenitor cells in SVZ were observed using immunofluorescent double labeling of either NG2 (marker of progenitor cells) and 5-bromodeoxyuridine (BrdU), or O4 (marker of OL precursor cells) and BrdU. RESULTS: All rats in the PVL group manifested either mild or severe white matter injury under light microscopy, and had higher pathological scores of white matter compared with the sham group at 7 and 21 days after PVL (P<0.05). Electron microscopy showed that the number and thickness of myelin sheath in the PVL group were significantly reduced compared with the sham group (P<0.01). O4-positive OL precursor cells in the white matter observed under fluorescence microscopy were significantly reduced in the PVL group compared with the sham group (P<0.05). BrdU/NG2-positive cells in the SVZ increased significantly in the PVL group 48 hours after PVL and migrated into the periventricular area, reaching a peak on day 7 after PVL. BrdU/O4-positive newborn cells began to appear in the periventricular area 72 hours after PVL, and the number of BrdU/O4-positive cells in the PVL group was statistically more than in the sham group on day 21 after PVL (P<0.05). CONCLUSIONS: Ischemia may induce brain self-repair in neonatal rats, resulting in activation and proliferation of NG2 glial progenitor cells in the SVZ migration and differentiation into OL precursor cells in periventricular white matter.