Enhancement of musculocutaneous nerve reinnervation after vascular endothelial growth factor (VEGF) gene therapy

Abstract

Background: Vascular endothelial growth factor (VEGF) is not only a potent angiogenic factor but it also promotes axonal outgrowth and proliferation of Schwann cells. The aim of the present study was to quantitatively assess reinnervation of musculocutaneous nerve (MCN) stumps using motor and primary sensory neurons after plasmid phVEGF transfection and end-to-end (ETE) or end-to-side (ETS) neurorrhaphy. The distal stump of rat transected MCN, was transfected with plasmid phVEGF, plasmid alone or treated with vehiculum and reinnervated following ETE or ETS neurorrhaphy for 2 months. The number of motor and dorsal root ganglia neurons reinnervating the MCN stump was estimated following their retrograde labeling with Fluoro-Ruby and Fluoro-Emerald. Reinnervation of the MCN stumps was assessed based on density, diameter and myelin sheath thickness of regenerated axons, grooming test and the wet weight index of the biceps brachii muscles.

Results: Immunohistochemical detection under the same conditions revealed increased VEGF in the Schwann cells of the MCN stumps transfected with the plasmid phVEGF, as opposed to control stumps transfected with only the plasmid or treated with vehiculum. The MCN stumps transfected with the plasmid phVEGF were reinnervated by moderately higher numbers of motor and sensory neurons after ETE neurorrhaphy compared with control stumps. However, morphometric quality of myelinated axons, grooming test and the wet weight index were significantly better in the MCN plasmid phVEGF transfected stumps. The ETS neurorrhaphy of the MCN plasmid phVEGF transfected stumps in comparison with control stumps resulted in significant elevation of motor and sensory neurons that reinnervated the MCN. Especially noteworthy was the increased numbers of neurons that sent out collateral sprouts into the MCN stumps. Similarly to ETE neurorrhaphy, phVEGF transfection resulted in significantly higher morphometric quality of myelinated axons, behavioral test and the wet weight index of the biceps brachii muscles.

Conclusion: Our results showed that plasmid phVEGF transfection of MCN stumps could induce an increase in VEGF protein in Schwann cells, which resulted in higher quality axon reinnervation after both ETE and ETS neurorrhaphy. This was also associated with a better wet weight biceps brachii muscle index and functional tests than in control rats.

Figure 1

Immunofluorescence staining for VEGF. Immunofluorescence staining for VEGF in the cryostat sections through intact musculocutaneous nerve (A) and distal stump of MCN transfected with plasmid phVEGF 2 months after its end-to-end (B) and end-to-side (C) neurorrhaphy. Double immunostaining for VEGF (Alexa-488) and neurofilaments (TRITC) indicated increased VEGF staining of Schwann cells in distal stump of MCN transfected with plasmid phVEGF 2 months after its end-to-end neurorrhaphy (D). Moreover, double immunostaining for VEGF (Alexa-488) and RECA (TRITC) displayed location of VEGF protein in blood vessels of transfected MCN stump 2 months after its end-to-side neurorrhaphy (E). Arrows - Schwann cells; arrowheads - blood vessels. Scale bars for A-C = 90 μm, D = 20 μm, E = 30 μm, for insets = 3 μm

Figure 2

Representative sections of C8 dorsal root ganglion and spinal cord segment.A. A schematic drawing of double retrograde labeling of neurons following end-to-side neurorrhaphy of MCN stump with UN. FE – Fluoro-Emerald, FR – Fluoro-Ruby. B,C. Representative cryostat sections of C8 dorsal root ganglion (B) and C8 spinal cord segment (C) from rat 2 months after ETS neurorrhaphy of the MCN stump transfected with plasmid phVEGF illustrating retrograde labeled primary sensory and motor neurons. Red fluorescence labeled neurons with axons present only in the UN distal to ETS neurorrhaphy. Green fluorescence loaded neurons whose axons were damaged during surgical treatment and regenerated only into the MCN distal stump. Yellow fluorescence of labeled neurons (arrows) resulted from a mixture of red and green fluorescence indicating the neurons, the donor axons of which were present in the UN that had sent out collateral sprouts into the distal stump of the MCN. Scale bars = 100 μm

Figure 3

Representative semi-thin sections through MCN. Representative transverse semi-thin sections through the MCN of intact rat (A) and MCN stump transfected with plasmid phVEGF (B, D) or plasmid alone (C, E) 2 months after their end-to-end (ETE) and end-to-side (ETS) neurorrhaphy. Scale bars = 2 μm

Figure 4

Distribution analyses of myelinated axon diameters. Distribution analyses of myelinated axon diameters in MCN stumps transfected with plasmid phVEGF and plasmid alone 2 months after their end-to-end and end-to-side neurorrhaphy. In comparison to transfection with plasmid alone, the phVEGF transfection induced a decreased percentage (↓) of myelinated axons with diameters from 1.1 to 2.0 μm, while there was an increased percentage (↑) in the range of 2.1 to 8.0 μm and 2.1 to 7.0 μm after end-to-end and end-to-side neurorrhaphy, respectively