Global expression of NGF promotes sympathetic axonal growth in CNS white matter but does not alter its parallel orientation

Abstract

Axonal regeneration is normally limited after injuries to CNS white matter. Infusion of neurotrophins has been successful in promoting regenerative growth through injured white matter but this growth generally fails to extend beyond the infusion site. These observations are consistent with a chemotropic effect of these factors on axonal growth and support the prevailing view that neurotrophin-induced axonal regeneration requires the use of gradients, i.e., gradually increasing neurotrophin levels along the target fiber tract. To examine the potential of global overexpression of neurotrophins to promote, and/or modify the orientation of, regenerative axonal growth within white matter, we grafted nerve growth factor (NGF) responsive neurons into the corpus callosum of transgenic mice overexpressing NGF throughout the CNS under control of the promoter for glial fibrillary acidic protein. One week later, glial fibrillary acidic protein and chondroitin sulfate proteoglycan immunoreactivity increased within injured white matter around the grafts. NGF levels were significantly higher in the brains of transgenic compared with non-transgenic mice and further elevated within injury sites compared with the homotypic region of the non-injured side. Although there was minimal outgrowth from neurons grafted into non-transgenic mice, extensive parallel axonal regeneration had occurred within the corpus callosum up to 1.5 mm beyond the astrogliotic scar (the site of maximum NGF expression) in transgenic mice. These results demonstrate that global overexpression of neurotrophins does not override the constraints limiting regenerative growth to parallel orientations and suggest that such factors need not be presented as positive gradients to promote axonal regeneration within white matter.

Fig. 1

GFAP-IR is increased near parasagittal knife lesions. (A) Cresyl violet-stained section of brain from a GFAP-NGF-Tg mouse having undergone a parasagittal knife lesion (the plane of knife entry is indicated by the arrow) 1 mm to the right of midline. The injured hemisphere (right side of micrograph) exhibited a furrow in the neocortex (CTX) encroaching on the corpus callosum (cc). Extensive neuronal loss occurred in the 300-μm wide cortical zone medial to the injury. Small cells, likely phagocytes, infiltrated the corpus callosum within the zone of injury. (B) Low power micrograph of a brain section from a non-transgenic mouse. The injured hemisphere (right) showed markedly increased GFAP-IR in all regions, including the corpus callosum and the overlying neocortex, compared with the uninjured (left) side. (C, D) High power micrographs centered at the apices of the lateral ventricles (LV) of the injured non-transgenic mouse shown in panel B. Astrocytes appeared hypertrophic in all areas on the injured side (panel D) compared with the corresponding contralateral field (panel C). (E) Low power micrograph showing a section from a GFAP-NGF-Tg mouse that had undergone knife lesion in the same manner. Increased GFAP-IR was evident on the injured side (right) similar to that in the non-transgenic mouse. (F, G) High power images of the injured (panel G) and uninjured (panel F) sides of the GFAP-NGF-Tg mouse section shown in panel E. Hc, hippocampus. Scale bars=500 μm in panels A, B, E; 150 μm in panels C, D, F, G.

Fig. 2

Quantification of GFAP-IR and NGF protein in knife-lesioned mice. (A) The illustration shows regions in cross-section (dark gray areas enclosed in dashed lines) in which GFAP-IR was quantified on the side ipsilateral (I) and contralateral (C) to the knife lesion (black). This same illustration shows the blocks of tissue (dark gray volumes enclosed in dashed lines) sampled for measurement of NGF protein using ELISA. (B) GFAP-IR is quantified as percent of tissue area that is GFAP-immunoreactive. GFAP-IR was significantly higher on the injured side (I) of both non-transgenic (white) and GFAP-NGF-Tg (black) mice compared with the homotypic region on the contralateral side (C). GFAP-IR was not significantly different between non-transgenic and GFAPNGF-Tg mice. (C) NGF protein was significantly higher in tissue sampled from GFAP-NGF-Tg mouse brains compared with non-transgenic mouse brains irrespective of injury. NGF was further elevated (*p<0.05) on the injured side (I) compared with the uninjured side (C) in GFAP-NGF-Tg mice. Bars represent mean±SEM.

Fig. 3

Viable grafts are clearly distinguishable from non-viable grafts. (A) A non-viable sympathetic graft (G) is shown placed within the corpus callosum (cc) stained using the SPG method. The graft showed predominantly yellow autofluorescence and contained several cystic cavities (*). In contrast, the caudate-putamen (CPu) was stained blue due to dense dopaminergic innervation. (B) A section adjacent to panel A stained with cresyl violet. As is the case with SPG staining, cystic cavities (*) were evident but no neurons could be seen. Instead, the graft was replete with small cells, likely to be phagocytes. (C) A viable sympathetic graft (G), stained blue using the SPG method, located within the lateral ventricle (LV) of a GFAP-NGF-Tg mouse (NGF/NGF). A dense plexus of blue fibers was found within the graft as well as along both ventricular walls. (D) A section adjacent to panel C showing the typically intense GFAP-IR within the subependyma, as well as elevated GFAP-IR anterior to the graft (arrow). CTX, neocortex. Scale bars=200 μm in panel A; 150 μm in panels B-D.

Fig. 4

Representative images of grafted ganglia stained with cresyl violet (panel labels: donor/host). Sympathetic neuronal profiles could be identified based on size and morphology, and generally appeared in isolated clusters (arrows) near the perimeters of grafts. Scale bars=200 μm.

Fig. 5

Quantification of neurons within grafted sympathetic ganglia stained with cresyl violet. (A) Graft area was significantly greater in ganglia harvested from GFAP-NGF-Tg mice compared with those harvested from non-transgenic mice (p=0.001). No significant host effect on graft area was detected (p=0.11; host × donor, p=0.32). (B) Significantly more neurons were counted per section in GFAP-NGF-Tg hosts compared with non-transgenic hosts (p=0.02). The donor effect was not significant (p=0.99; host × donor, p=0.06). (C) Neuronal density was significantly greater in GFAP-NGF-Tg hosts (p=0.02). No significant donor effect on neuronal density was detected (p=0.09; host × donor, p=0.12). Bars represent mean±SEM.

Fig. 6

Sympathetic grafts (G) placed in both GFAP-NGF-Tg and non-transgenic brains were associated with increased GFAP- and CSPG-IR in the host. (A) An SPG-stained, non-transgenic sympathetic graft placed in the corpus callosum (cc) of a non-transgenic mouse. The graft contained many fibers but only one could be seen exiting the graft, extending a short distance into the corpus callosum (arrow). (B) An adjacent section through the graft shown in panel A immunostained for GFAP. As demonstrated here, GFAP-IR is rarely observed within grafts but increased GFAP-IR surrounding the graft was always evident. (C) An adjacent section through the graft shown in panel A immunostained for CSPG. CSPG-IR is generally greater in the cc compared with the overlying cortex (CTX) and the graft interior and is further elevated in patches within the cc on either side of the graft (arrowheads). (D) An SPG-stained graft harvested from a GFAP-NGF-Tg mouse placed in the corpus callosum of a GFAP-NGF-Tg mouse. (E) Section adjacent to panel D showing elevation of GFAP-IR around the graft. GFAP-immunoreactive cells (arrowhead) were seen within the graft in this case, though this observation did not generalize to this (NGF/NGF) or any experimental group. (F) As in panel C, CSPG-IR is elevated in patches on either side of the graft. LV, lateral ventricle. Scale bars=200 μm.

Fig. 7

Sections stained using the SPG method showing axonal growth from grafts (G) placed within the corpus callosum (cc). In all panels, the direction lateral to the grafts is to the right. (A) Wt/Wt case showing a few fibers (arrows) extending within the cc (borders of the fiber tract are indicated by arrowheads) in parallel with the fiber tract. These fibers were mostly limited to a yellow autofluorescent portion of the tract, characteristic of injured tissue. (B) Wt/Wt case with fibers (arrow) shown extending primarily within a yellow autofluorescent portion of the cc in directions orthogonal to the fiber tract. Representative NGF/Wt and NGF/NGF cases with similar placement within the cc are shown in panels C and G. High power micrographs of the fiber tracts lateral to the grafts are shown in panels D-F and H-J, respectively. Though SPG+ axons are apparent within the NGF/Wt graft shown in panel C, no outgrowth was observed either medial to the graft or 0.4 mm (panel D), 0.8 mm (panel E) or 1.2 mm (panel F) lateral to the graft. In contrast, a dense plexus of fibers extended laterally within the cc from the NGF/NGF graft shown in panel G. Outgrowth dorsal to the graft (arrow) within an injured yellow autofluorescent region is mixed in orientation, whereas growth that is more distal was essentially parallel to the fiber tract at 0.4 mm (panel H), 0.8 mm (panel I) or 1.2 mm (panel J) lateral to the graft. (K) A section adjacent to that shown in panel G shows laminin immunoreactivity primarily associated with the choroid plexus (*), the graft itself and the vasculature (e.g., arrows). Comparison with the pattern of axonal outgrowth in panel G suggests that it is unlikely that this outgrowth occurs preferentially along laminin-rich vessels. Scale bars=200 μm in panels A-C, G, K; 100 μm in panels D-F, H-J.

Fig. 8

Sections stained using the SPG method showing axonal growth from grafts (G) placed within the corpus callosum (cc) of GFAP-NGF-Tg mice. In all panels, the direction lateral to the grafts is to the right. (A) NGF/NGF case with outgrowth extending within the cc (borders of the fiber tract indicated by arrowheads) in both medial and lateral directions. These axons extended beyond the zones of increased GFAP-IR surrounding the graft (cf. adjacent section shown in panel B). (C) Wt/NGF case with outgrowth that extended beyond the midline (note pericallosal artery, a). High power micrographs show axons that have extended 0.6 mm beyond the midline (panel D) and through the midline (panel E). These axons appear to be extending predominantly in parallel with the fiber tract. LV, lateral ventricle. Scale bars=200 μm in panels A-C; 100 μm in panels D, E.

Fig. 9

Axonal growth in the corpus callosum of GFAP-NGF-Tg mice is longer than that in non-transgenic mice (p<0.0001). The longest outgrowth was measured from non-transgenic grafts placed in the corpus callosum of GFAP-NGF-Tg mice (Wt/NGF), followed by grafts harvested from GFAP-NGF-Tg mice placed in GFAP-NGF-Tg mice (NGF/NGF). Both groups utilizing nontransgenic hosts showed minimal growth in the corpus callosum. Bars represent mean±SEM.