Article Figures & Data
Figures
- Fig. 1.
Postnatal expression of HSP27 protein in the DRG and expression after sciatic nerve injury. A, At P2, HSP27 immunoreactivity is detectable in a subpopulation of neurons in the L4 DRG of naive animals. B, Two days after nerve injury at P0, the intensity of HSP27 immunoreactivity increases dramatically in the ipsilateral (ipsi) DRG; however, the number of cells that are positive does not increase (see Results).C, The number of DRG neurons constitutively immunoreactive for HSP27 increases postnatally. Numbers are the mean ± SD from three or four animals per age group.contra, Contralateral. Scale bars, 50 μm.
- Fig. 2.
Most injured DRG neurons die after P0 axotomy, but the number of HSP27-immunoreactive neurons does not change.A, After nerve injury at P0, there is a dramatic loss of neurons in the L4 DRG by P7. C, However, the number of neurons expressing HSP27 does not decrease (n = 4–5 per group). B, Not all DRG neurons project into the sciatic nerve (schematic); therefore not all DRG neurons are axotomized by the sciatic nerve transection. ax., Axotomized.
- Fig. 3.
DRG neurons that survive P0 sciatic nerve section express HSP27. A, Seven days after P0 sciatic nerve section, axotomized neurons in the L4 DRG were identified by immunohistochemistry for c-jun. B, Double-labeling for HSP27 reveals that all DRG neurons with distinct nuclear c-jun labeling are also HSP27 immunoreactive.
- Fig. 4.
Most HSP27-immunoreactive neurons do not undergo apoptosis after P0 axotomy. A, A section of spinal cord with attached L4 DRG was double-labeled for HSP27 immunoreactivity (red/orange) and TUNEL (green) 48 hr after P0 sciatic nerve transection. The arrow indicates a single double-labeled cell that appears yellow. B, C, Sections of DRG were double-labeled for neurofilament (B) and TUNEL (C), demonstrating that most TUNEL-positive cells are also neurofilament immunoreactive (arrows).
- Fig. 5.
HSP27 expression correlates with survival after NGF withdrawal in vitro. Dissociated cultures from P0 lumbar DRG were cultured for 48 hr in the presence (d) or absence (a–c) of NGF.a–c, The same field is shown, immunostained for PGP9.5 to identify neurons (a), HSP27 (b), or Hoechst 33342 to visualize nuclear morphology (c). After 48 hr without NGF, neurons that expressed HSP27 had round healthy nuclei (arrows ina–c), whereas the majority of those that were negative for HSP27 immunoreactivity had pyknotic nuclei (arrowheads in a–c). e, The percentage of HSP27-immunopositive neurons with pyknotic nuclei was counted, and the same was done for HSP27-immunonegative neurons. Data shown are means ± SD from four independent experiments; a total of 5533 neurons were counted. d, In the presence of NGF, HSP27 was not necessary for either survival or neurite outgrowth (darkcells are HSP27 positive). Scale bars, 50 μm.
- Fig. 6.
Expression of HSP27 in dissociated neonatal DRG neurons reduces nuclear pyknosis and increases survival after NGF withdrawal. a, b, P0 DRG cultures were infected with adenovirus (Ad) expressing human HSP27 (a) or β-galactosidase as a control (b). Forty hours later, NGF was withdrawn by refeeding with medium containing anti-NGF (1:250). Cultures were fixed 48 hr later and double immunostained with the neuron-specific antibody PGP9.5 and either anti-human HSP27 (a) or anti-β-galactosidase (-β-gal; b). The antibody to human HSP27 does not recognize rat HSP27, so it labels only infected cells. c, d, Nuclear morphology was visualized with Hoechst 33342. Arrows in a–dindicate neurons. e, The percentages of nlacZ- or HSP27-immunoreactive cells with pyknotic nuclei in the presence and absence of NGF were determined. Data shown are the means ± SEM of triplicates from a single experiment (200 cells were counted for each replicate). The experiment was repeated four times with similar results. f, To assess neuronal survival, total surviving neurons were counted in a 50 mm2 area of each well at the indicated times (in hours) after NGF withdrawal; typically, this area contained 150–200 neurons at the time of NGF withdrawal. Data shown are the means ± SEM of triplicates from a single experiment. The experiment was repeated three times with similar results.
- Fig. 7.
Expression of HSP27 in dissociated cultures of SCG neurons reduces nuclear pyknosis and increases survival after NGF withdrawal. Neuronal cultures of neonatal SCG were infected with adenovirus expressing human HSP27 or β-galactosidase as a control. Forty-eight hours later, NGF was withdrawn. A, After 24 hr, cells were fixed and immunostained for human HSP27 or β-galactosidase, and nuclear morphology was visualized with Hoechst 33342. HSP27-expressing neurons had far fewer pyknotic nuclei than did β-galactosidase-expressing control neurons. Data shown are means ± SEM of triplicates from a single experiment that was repeated twice with similar results. B, To assess neuronal survival, MTT assays were done 48 hr after NGF withdrawal from cultures infected with either human HSP27- or β-galactosidase-expressing adenoviruses at various m.o.i. values. Data shown are the means ± SEM of triplicates from a single experiment. Similar experiments were done three times with similar results.
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