Dynamic pattern of reg-2 expression in rat sensory neurons after peripheral nerve injury

Abstract

The 16 kDa pancreatitis-associated protein Reg-2 has recently been shown to facilitate the regeneration of motor and sensory neurons after peripheral nerve injury in the adult rat. Reg-2 has also been shown to be a neurotrophic factor that is an essential intermediate in the pathways through which CNTF supports the survival of motor neurons during development. Here we report the dynamic expression of Reg-2 in rat sensory neurons after peripheral nerve injury. Reg-2 is normally not expressed by dorsal root ganglion (DRG) cells, but we show, using immunocytochemistry, that Reg-2 is rapidly upregulated in DRG cells after sciatic nerve transection and after 24 hr recovery is expressed almost exclusively in small-diameter neurons that bind the lectin Griffonia simplicifolia IB4 and express the purinoceptor P2X3. However, by 7 d after axotomy, Reg-2 is expressed in medium to large neurons and coexists partly with the neuropeptides galanin and neuropeptide Y, which are also upregulated after peripheral nerve transection. At this time point, Reg-2 is no longer expressed in small neurons, and there is no colocalization with IB4 binding neurons, demonstrating a shift in Reg-2 expression from one subset of DRG neurons to another. We also show by double labeling for activating transcription factor 3, a transcription factor that is upregulated after nerve injury, that Reg-2 expression occurs predominantly in axotomized DRG cells but that a small percentage of uninjured DRG cells also upregulate Reg-2. The selective expression within IB4/P2X3 cells, and the dynamic shift from small to large cells, is unique among DRG peptides and suggests that Reg-2 has a distinctive role in the injury response.

Fig. 1.

Expression of Reg-2 in DRG after peripheral nerve injury. A–D show Reg-2 immunoreactivity in ipsilateral (A–C) and naïve control (D) lumbar DRG 1 d (A), 7 d (B), and 30 d (C) after sciatic nerve transection. Reg-2 is upregulated after sciatic transection, but expression is dynamic, appearing predominantly in small cells at 1 d (A) but in medium and large cells at 7 d (B). E–H show the disposition of Reg-2-immunoreactive axons in an L4 ganglion (DRG) with attached ventral root (VR), dorsal root (DR), and spinal nerve (SN) at 1 d after sciatic transection. The labeling in Eshows the areas that were sampled for the high-magnification images inF–H. Immunoreactive axons (arrows) can be observed within and on the peripheral side of the ganglion (F) as well as within the spinal nerve (G). However, very few axons were present on the central side of the ganglion or within the attached dorsal root (H) or ventral root. Scale bars:A–D, 100 μm ; E, 200 μm;F–H, 50 μm.

Fig. 2.

Size distribution of Reg-2-immunoreactive sensory neurons in L4/5 DRG after sciatic nerve transection. Note that at 24 hr many small-diameter sensory neurons are immunoreactive, but by 5 d predominantly medium-diameter cells show Reg-2 expression. At 5 d after transection, some neurons are large, as indicated by thelong tail seen on the size distribution graph, which is not seen by 8 weeks after transection.

Fig. 3.

Analysis of Reg-2 immunoreactivity in the spinal cord and brain stem. A–D show the lumbar spinal cord 7 d after sciatic nerve transection. Reg-2 immunoreactivity is expressed in the ventral horn (VH) in axotomized motoneurons (A, arrows) but is absent from the dorsal horn (DH), indicating that it is not present in the central terminals of axotomized primary afferents.B–D show the superficial dorsal horn at high magnification, stained for IB4, Reg-2, or CGRP.Asterisks indicate the central terminal field of the axotomized sciatic nerve, which has downregulated IB4 (B). However, Reg-2 immunoreactivity in this region is not above background staining (C), although CGRP staining confirms that primary afferent terminals are present (D). E andF show the dorsal medulla 30 d after sciatic nerve transection. Ipsilateral to the transection, neuropeptide Y (NPY) immunoreactivity is upregulated within the gracile nucleus (E) in the central terminal fields of the axotomized primary afferents (asterisk). However, there is no indication of Reg-2 immunoreactivity in that region (F). Scale bars: A, 200 μm; B–F, 100 μm.

Fig. 4.

Axonal transport of Reg-2. A,C, and E show anterograde accumulation proximal to a L4/L5 dorsal root ligature, and B,D, and F show anterograde accumulation proximal to a sciatic nerve ligature. Vertical arrows inA and B indicate the site of each ligature. CGRP shows a prominent accumulation in dorsal roots (A) and in sciatic nerve (B), whereas very little Reg-2 accumulation (C, D, arrows) is seen in dorsal roots compared with sciatic nerve. IB4 staining proximal to the ligatures is not as prominent as CGRP but is present in both dorsal roots (E) and sciatic nerve (F). Scale bars: A,C, E, 100 μm; B,D, F, 200 μm.

Fig. 5.

Colocalization of Reg-2-positive cells 24 hr after sciatic nerve transection. Immunofluorescent staining of single L5 DRG sections using Reg-2 (A, C,E) and IB4-FITC conjugate (B) or P2X₃ (D) or trkA (F) polyclonal antibodies 24 hr after sciatic nerve transection. Arrows show double-labeled cells, andarrowheads show Reg-2-positive cells that are negative for the second marker. Note that many Reg-2-positive cells show IB4 or P2X3 labeling. Scale bar, 50 μm.

Fig. 6.

Colocalization of Reg-2-positive cells 7 d after sciatic nerve transection. Immunofluorescent staining of single L4/5 DRG sections using anti-Reg-2 polyclonal antibody (A, C, E) and IB4-FITC conjugate (B) or galanin (D) or NPY (F) polyclonal antibodies, 7 d after sciatic nerve transection.Arrows show double-labeled cells, andarrowheads show Reg-2-positive cells that are negative for the second marker. Note that many Reg-2-positive cells at this time point show colocalization with NPY and galanin but not IB4. Scale bar, 50 μm.

Fig. 7.

Colocalization of Reg-2-positive profiles with axotomized sciatic projecting neurons. Immunofluorescent staining of single L5 DRG sections using Reg-2 (A, C) and ATF3 (B, D) polyclonal antibodies 24 hr after sciatic nerve transection is shown. Arrows inC and D show profiles that are double labeled for Reg-2 and ATF3. Note that after axotomy, a small number of Reg-2-positive profiles do not colocalize with the injured nerve marker ATF3 (arrowheads). Scale bars, 50 μm.