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. 2003 Apr 1;23(7):3028-38.
doi: 10.1523/JNEUROSCI.23-07-03028.2003.

Increased sensitivity of injured and adjacent uninjured rat primary sensory neurons to exogenous tumor necrosis factor-alpha after spinal nerve ligation

Maria Schäfers  1 , Doo H Lee, Dominik Brors, Tony L Yaksh, Linda S Sorkin
Affiliations

Increased sensitivity of injured and adjacent uninjured rat primary sensory neurons to exogenous tumor necrosis factor-alpha after spinal nerve ligation

Maria Schäfers et al. J Neurosci. .

Abstract

Tumor necrosis factor-alpha (TNF) is upregulated after nerve injury, causes pain on injection, and its blockade reduces pain behavior resulting from nerve injury; thus it is strongly implicated in neuropathic pain. We investigated responses of intact and nerve-injured dorsal root ganglia (DRG) neurons to locally applied TNF using parallel in vivo and in vitro paradigms. In vivo, TNF (0.1-10 pg/ml) or vehicle was injected into L5 DRG in naive rats and in rats that had received L5 and L6 spinal nerve ligation (SNL) immediately before injection. In naive rats, TNF, but not vehicle, elicited long-lasting allodynia. In SNL rats, subthreshold doses of TNF synergized with nerve injury to elicit faster onset of allodynia and spontaneous pain behavior. Tactile allodynia was present in both injured and adjacent uninjured (L4) dermatomes. Preemptive treatment with the TNF antagonist etanercept reduced SNL-induced allodynia by almost 50%. In vitro, the electrophysiological responses of naive, SNL-injured, or adjacent uninjured DRG to TNF (0.1-1000 pg/ml) were assessed by single-fiber recordings of teased dorsal root microfilaments. In vitro perfusion of TNF (100-1000 pg/ml) to naive DRG evoked short-lasting neuronal discharges. In injured DRG, TNF, at much lower concentrations, elicited earlier onset, markedly higher, and longer-lasting discharges. TNF concentrations that were subthreshold in naive DRG also elicited high-frequency discharges when applied to uninjured, adjacent DRG. We conclude that injured and adjacent uninjured DRG neurons are sensitized to TNF after SNL. Sensitization to endogenous TNF may be essential for the development and maintenance of neuropathic pain.

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Figures

Fig. 1.
Fig. 1.
Extracellular dorsal root single fiber recording.A, Schematic of the preparation. The ganglion is suspended in the middle chamber; the dorsal root and spinal nerve are in adjacent mineral oil-filled chambers for recording (R) and stimulation (S), respectively. B, Electrical stimulation of the spinal nerve evoked two fixed waveforms. Templates (dotted line) were created by computer-based analysis of the waveforms and amplitudes of the evoked spikes (Spikes 2). C, After electrical stimulation, activity was recorded from the microfilament while the DRG was perfused first with ACSF and then with exogenous TNF (here, 100 pg/ml). Computer-based matching of the electrically evoked templates to the spikes (continuous line) obtained during the course of baseline, TNF perfusion, and washout allowed information on separate fibers to be collected simultaneously. Activity from each fiber was extracted and processed separately; the response of the A-δ fiber in B is illustrated; bin width in this example is 1.25 min.
Fig. 2.
Fig. 2.
Mechanical withdrawal thresholds after intraganglionic injection of TNF obtained from testing sites (x) in L5, L4, and L3 dermatomes. In naive rats (left), intraganglionic TNF injection at 10 pg/ml, but not 0.1 pg/ml, reduced mechanical withdrawal thresholds tested in L5 and L4 but not in L3 dermatomes. In rats with SNL and intraganglionic injection (right) of saline, thresholds were significantly reduced after day 8 in L5 and L4 but not in L3 dermatomes. Addition of SNL and intraganglionic TNF at the previous subthreshold dose of 0.1 pg/ml elicited a faster onset allodynia that was maximal at day 1 in the L5 and L4 dermatomes (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0005 vs saline- or saline + SNL-treated group; one-way ANOVA with Tukeypost hoc test).
Fig. 3.
Fig. 3.
Changes in weight gain, duration of spontaneous paw elevation, and spontaneous limb movement were measured as potential indices of ongoing pain. A, Rats with SNL and intraganglionic injection of TNF, but not other treatment groups, showed no increase of weight when weighed 4 d after surgery. Weight increases were comparable with other treatment groups during days 4–8 and 8–10. B, Cumulative duration of spontaneous paw elevation was counted over a 5 min period. Rats with SNL and TNF injection kept their paws elevated for more than half of the time after day 4. C, Locomotor activity was assessed by analysis of spontaneous movement of the ipsilateral paw. No differences were found between the treatment groups (**p < 0.005, ***p < 0.001 vs SNL + saline-treated group; one-way ANOVA with Tukey post hoc test).
Fig. 4.
Fig. 4.
Propidium iodine exclusion staining was used to assess the induction of cell death by intraganglionic injections.A, Rats with in vivo L5 intraganglionic injection of TNF (10 pg/ml), but not saline, BSA (10 pg/ml), or methanol, displayed markedly lowered von Frey thresholds on days 3 and 6 after injection. B, DRG from rats with in vivo injection of methanol, but not TNF, BSA, or saline, showed increased numbers of dead DRG cells. Ganglia were collected 6 d after in vivo injection (*p < 0.05, **p < 0.005 vs saline-treated group; one-way ANOVA with Tukey post hoc test).
Fig. 5.
Fig. 5.
Mechanical withdrawal thresholds tested in the L4 dermatome after SNL and preemptive systemic treatment with saline or etanercept. Treatment was started 2 d before surgery after the first baseline testing and repeated on postoperative days 0, 3, 6, 9, and 12 (arrows). Rats with SNL and saline developed a sustained mechanical allodynia that was present from day 1 until the end of the experiment. In rats with SNL and etanercept, mechanical withdrawal thresholds, although reduced from baseline, were significantly higher than in saline-treated animals (*p < 0.05 vs SNL + saline-treated group; one-way ANOVA with Tukey post hoc test).
Fig. 6.
Fig. 6.
Dose–response curves of TNF-evoked discharges. The contralateral normal (L5R), ipsilateral injured (L5L), and ipsilateral adjacent uninjured (L4L) DRG were superfused with five consecutively increasing TNF doses while activity was recorded from dorsal root microfilaments in vitro. TNF applications were separated by washout periods. A, Peak response. Units from L5R DRG were silent at baseline and required a relatively high (100 pg/ml) TNF dose to evoke firing over 1 Hz. In units of injured L5L and adjacent uninjured L4L DRG, lower TNF doses (0.1–10 pg/ml) were sufficient to elicit higher frequency discharges.B, Duration of TNF-evoked response. In ipsilateral injured (L5L) but not in adjacent (L4L) DRG, TNF-evoked responses persisted longer than those seen in normal DRG at lower concentrations. At the highest TNF concentration, the duration of TNF-evoked responses was longer in L5R DRG than in either L5L or L4L DRG (**p < 0.005, ***p < 0.001 vs L5R DRG; Kruskal–Wallis test with Dunn post hoc test;+++p < 0.001 vs L5R response at 0.1 pg/ml TNF; Friedman test with Dunn post hoc test).
Fig. 7.
Fig. 7.
Comparison of TNF-evoked responses in injured (L5L) DRG to sequential perfusion with the same TNF dose [REP (1 pg/ml)] or consecutively ascending TNF doses [ASC (0.1–1000 pg/ml)]. A, Perfusions with the first three ascending TNF doses (ASC,I–III) resulted in ascending peak responses; this reached significance during the third perfusion. Peak response discharge did not change across the first three perfusions with the same TNF concentration (REP,I–III). The fourth and fifth TNF perfusion elicited a lower peak discharge rate for both series. B, Over repetitive DRG perfusions with the same TNF dose, latencies to peak responses did not change. In contrast, latencies dose-dependently decreased with sequential ascending TNF doses (+ p < 0.05,++ p < 0.005,+++ p < 0.001 vs response at interval I; Friedman test with Dunn post hoc test; ***p < 0.001, ****p < 0.0005 REP vs ASC; Mann–Whitney test).
Fig. 8.
Fig. 8.
Peristimulus histograms of mean TNF-evoked discharge rates of all fibers that were initially not spontaneously active (SPA−) from contralateral intact (Intact DRG: L5R, left), ipsilateral injured (Injured DRG: L5L, middle), and adjacent injured (Adjacent DRG: L4L, right) DRG after SNL. All DRG were perfused with 1 pg/ml TNF (shaded area). Note: data for L5R DRG are magnified 10× compared with L5L and L4L DRG. Left, Latencies to evoked peak discharges and peak responses are similar among all three fiber types of L5R DRG, but C fibers display a longer duration TNF response. Middle, In injured L5L DRG, TNF-evoked peak discharge rates were highest in A-β and C fibers and markedly higher than those observed in intact DRG. Additionally, latencies to TNF-evoked peak discharges were shorter in fibers of injured compared with those of intact DRG.Right, In all fiber types of adjacent L4L DRG, TNF-evoked peak discharge rates were markedly higher than those observed in intact DRG. TNF+ = TNF responsive.
Fig. 9.
Fig. 9.
Peristimulus histograms of mean TNF-evoked discharge rates of all spontaneously active (SPA+) fibers of L5L and L4L DRG after SNL. Both DRG were superfused with 1 pg/ml TNF (shaded area). Left, In injured L5L DRG, TNF increased firing rates mostly in A-δ fibers, whereas activity of A-β and C fibers was not changed from basal. Latencies for TNF-evoked peak discharge rates were markedly shorter than in normal DRG. Right, Compared with injured DRG, baseline firing rates were slightly lower in spontaneously active fibers of adjacent DRG. TNF evoked markedly higher peak responses in all fiber types of L4L DRG compared with fibers of both normal and SPA+ L5L injured DRG. TNF+ = TNF responsive.
Fig. 10.
Fig. 10.
Comparison of latencies to peak responses and duration and height of peak responses of TNF-responsive fibers among different fiber and DRG types. A, The latency to peak response is reduced in all A-β (A-beta)and A-δ (A-delta) fibers and spontaneously not active (SPA−) C fibers of injured (L5L) DRG. In adjacent (L4L) DRG, only SPA− C fibers have a reduced latency to the peak response. B, In normal (L5R) DRG, C fibers have a prolonged response to TNF-evoked discharges. In L5L DRG, the duration of TNF-evoked responses tends to be substantially prolonged in all A-β and A-δ fibers. In L4L DRG, SPA+ A-δ and SPA− A-β fibers also display a prolonged duration of TNF evoked responses. C, In L5R DRG, peak responses are low and not different between fiber types. In L5L DRG, all SPA− fibers and SPA+ A-δ fibers exhibit increased peak responses compared with L5R DRG. In L4L DRG, peak responses in all fiber types are even higher than in L5L DRG (*p < 0.05, **p < 0.005, ***p < 0.001 vs respective fiber type of L5R DRG; one-way ANOVA with Tukey post hoc test; peak responses for SPA+ fibers were calculated after background subtraction).
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