Perivascular cells increase expression of ciliary neurotrophic factor following partial denervation of the rat neurohypophysis

Abstract

The expression of ciliary neurotrophic factor (CNTF) was investigated immunocytochemically during the axonal degeneration and collateral axonal sprouting response that follows partial denervation of the rat neurohypophysis. A significant increase in the number of CNTF-immunoreactive (CNTF-ir) cells was observed in the neurohypophysis of partially denervated animals compared to age-matched sham-operated controls by 5 days post-denervation, remaining elevated throughout the 30 day post-denervation period. Stereometric assessment of the numbers of CNTF-ir cells within the partially denervated neurohypophysis demonstrated a 36% increase by 3 days following denervation reaching 130% of control values by 10 days post-lesion. The cell numbers remained elevated throughout the 30 day post-lesion period suggesting that CNTF may play a role in the neurosecretory axonal sprouting process known to occur between 10 and 30 days post-denervation. Subsequent preparations pairing anti-CNTF with antibodies against ED1, CR3, p75 low affinity neurotrophin receptor (p75(LNGFR)), and S100beta, demonstrated that CNTF was exclusively localized in a phenotypically-distinct population of perivascular cells. The association of perivascular cells with phagocytic activity was confirmed by dual-label fluorescence microscopy showing the colocalization of P75(LNGFR)-ir and OX-42-ir in cells expressing the ED-1 antigen. No increase in CNTF-ir was observed in non-injured animals in which heightened levels of neurosecretory activity were induced physiologically. These results suggest that increased CNTF-ir occurs in response to conditions which induce high levels of phagocytic activity by perivascular cells in the axotomized neurohypophysis which is sustained throughout a period in which axonal sprouting is known to occur in the partially denervated neurohypophysis.

Figure 1

Preparations pairing anti-CNTF with antibodies against CR3, p75 low affinity neurotrophin receptor (p75LNGFR), and S100β, demonstrated that CNTF was exclusively localized in a phenotypically-distinct population of perivascular cells. (A) Peroxidase labeling of CNTF in the partially denervated neurohypophysis revealed extensive CNTF-ir in small vellate cells (arrows) closely associated with blood vessels (v). (B) Dual fluorescence labeling showed no colocalization of CNTF-ir (white arrows, Cy-3) with the astrocyte marker S100β (asterisks, FITC). Note the extensive CNTF-ir processes and their close association with blood vessels. (C) Dual peroxidase staining demonstrated no colocalization of OX-42 (arrows, cobalt-DAB) with CNTF-ir (brown, peroxidase). (D–F) Dual fluorescent staining pairing anti-CNTF (D, arrows, FITC) with anti-P75^LNGFR (E, arrows, Cy-3), was consistently colocalized with P75^LNGFR-ir (F, arrows), although not all P75^LNGFR-ir cells were also CNTF-ir (F, asterisk) . (V=blood vessel). Mag bar = 20 uM.

Figure 2

The association of perivascular cells with phagocytic activity was confirmed by colocalization of CR3-ir and P75-ir in cells expressing the ED-1 antigen. (A) ED-1 profiles (arrows) increased by 3 days post-denervation and were localized within the cytoplasm of OX-42-ir microglial cells. (B) ED-1-ir was also localized within P75LNGFR-ir perivascular cells (arrows); (C) Occasionally, ED-1 was observed in CNTF-ir cells. (D) ED-1 (arrows) was never observed within S100β-ir pituicytes . (V=blood vessel). Mag bar = 20 uM.

Figure 3

Lesion-Induced Increase in CNTF-ir Perivascular Cells. Analysis of group differences (SPSS, V.9.0) confirmed that a significant increase in the number of CNTF-ir cells within the neurohypophysis occurred between 3 and 30 days following partial denervation (1 way ANOVA, F=2.775, df= 7, 49, P<.018). The increase in the number of CNTF-ir cells in lesioned animals compared to age-matched sham-operated controls was first significant at 5 days post-denervation and reached a peak at 10 days post-denervation. By 30 days following surgery the mean number of CNTF-ir cells was no longer significantly elevated. Time points and error bars represent the mean and standard error of the mean of 4–10 animals per group. PL indicates number of days post-lesion; Sh indicates age-matched sham control group.