Capsaicin-induced neuronal death and proliferation of the primary sensory neurons located in the nodose ganglia of adult rats

Abstract

To evaluate the potential for neuronal replacement following destruction of vagal afferent neurons, we examined nodose ganglia following i.p. capsaicin treatment of adult rats. Rats received capsaicin or vehicle followed by a regimen of 5'-bromo-2'-deoxyuridine injections (BrdU) to reveal DNA replication. Nodose ganglia were harvested at various times post-treatment and processed for 4',6-diamidino-2-phenylindole (DAPI) nuclear staining and immunofluorescence to estimate neuronal numbers and to determine vanilloid receptor, cleaved caspase 3, TUNEL, BrdU, the neuron-selective marker protein gene product (PGP) -9.5 and neurofilament-M-immunoreactivity. Twenty-four hours after capsaicin approximately 40% of nodose ganglion neurons expressed cleaved caspase 3-immunoreactivity and 16% revealed TUNEL staining, indicating that primary sensory neurons are killed by the capsaicin treatment of adult rats. The occurrence of neuronal death was confirmed by counts of DAPI-stained neuronal nuclei, which revealed >or=50% reduction of nodose neuron number by 30 days post-capsaicin. However, by 60 days post-capsaicin, the total numbers of neuronal nuclei in nodose ganglia from capsaicin-treated rats were not different from controls, suggesting that new neurons had been added to the nodose ganglia. Neuronal proliferation was confirmed by significant BrdU incorporation in nuclei of nodose ganglion cells immunoreactive for the neuron-specific antigen PGP-9.5 revealed 30 and 60 days post-capsaicin. Collectively, these observations suggest that in adult rats massive scale neurogenesis occurs in nodose ganglia following capsaicin-induced neuronal destruction. The adult nodose ganglion, therefore, provides a novel system for studying neural plasticity and adult neurogenesis after peripheral injury of primary sensory neurons.

Fig. 1

Decline and recovery of VR1-IR in NG after capsaicin treatment. Proportion of NG neurons expressing VR1-IR ± SEM (A; n=4 per time point per treatment). Immunofluorescence revealed VR1-IR in small and medium size neurons in the NG ganglia of vehicle- (B1) and capsaicin-treated rats (B2–3). However the proportion of VR1-IR neurons was decreased 3 days post-capsaicin (B2) but increased again by 30 days post-capsaicin (B3). Note, however, that the total number of NG neurons in capsaicin treated rats, as estimated by counts of neuronal nuclei (Fig. 5), was approximately 50% of the total for control. Therefore, the total number of VR1-IR neurons did not approach control levels until 60 days after capsaicin. Scale bars = 50 µm.

Fig. 2

Caspase-3-IR in NG neurons 24h after systemic treatment with capsaicin or vehicle (A; n = 4 per time point per treatment). Note the numerous cas-3-IR neurons in NG one day postcapsaicin treatment (B2; arrows). Very few NG neurons were cas-3-IR at any time point in vehicle-treated rats (B1) or capsaicin-treated rats 30 (B3) and 60 days post-treatment (arrows). Scale bars = 50 µm.

Fig. 3

Double immunofluorescence staining for TUNEL (red) and NfM (green) in the NG sections from capsaicin (A–C) and vehicle (D–F) treated rats. TUNEL positive glia cells were present in both the capsaicin (A) and vehicle (D) treated rats indicating physiological turnover previously reported in dorsal root ganglia after axotomy (McKay et al., 2002). ApoTome images showing examples of TUNEL labeled nuclei (A, C) from NfM immunoreactive neuron (B, C) in the plane X–Y (arrowhead). Optical planes, X–Z (top margin) and Y–Z (right margin), show the depth of this neuron (arrowheads). TUNEL-labeled nuclei were surrounded by NfM-immunoreactive cytoplasm in all three dimensions what indicates that dual labeled perikarya (arrowheads) of NG neurons were undergoing cell death (C, merged image). Images D–F show the example of TUNEL-negative (D) and NfM-immunoreactive (E) neuron that is not dying (arrowhead) from vehicle treated rat in all three planes (F, merged image). Scale bars = 50 µm.

Fig. 4

Double labeled sections of the NG for BrdU incorporation (A, D) and neuron-selective marker PGP-9.5 (B, E) from capsaicin (A–C) and vehicle (D–F) treated rats. Note that all BrdU-labeled neurons (A, arrowheads) were simultaneously PGP-9.5 immunoreactive (B, arrowheads) in capsaicin treated rats. Double labeling was confirmed on merged image analyzed in X–Y, X–Z and Y–Z optical planes revealing BrdU labeled nucleus surrounded by PGP-9.5 positive cytoplasm (C, arrowheads). Scale bars = 50 µm.

Fig. 5

Total numbers of DAPI-labeled neuronal nuclei in the left (upper graph) and the right (lower graph) NG from vehicle- and capsaicin-treated rats collected 3, 11, 30 and 60 days after treatment (n = 4 per time point per treatment). Bars represent average neuronal number ± SEM.

Fig. 6

Transmission electron micrographs transverse sections of cervical vagus, 180 days after systemic vehicle (A) or capsaicin injections (B). The majority of profiles are of medium and small unmyelinated fibers. Note the presence of very small axon profiles (arrows) in the tissue from the capsaicin-treated rat. These small profiles may be neurites extending from new neurons. Such small profiles never were detected in vehicle injected rats. Asterisk indicates myelinated fiber.