Mice lacking tPA, uPA, or plasminogen genes showed delayed functional recovery after sciatic nerve crush

Abstract

Axonal outgrowth during peripheral nerve regeneration relies on the ability of growth cones to traverse through an environment that has been altered structurally and along a basal lamina sheath to reinnervate synaptic targets. To promote migration, growth cones secrete proteases that are thought to dissolve cell-cell and cell-matrix adhesions. These proteases include the plasminogen activators (PAs), tissue PA (tPA) and urokinase PA (uPA), and their substrate, plasminogen. PA expression and secretion are upregulated in regenerating mammalian sensory neurons in culture. After sciatic nerve crush in mice, there was an induction of PA mRNAs in the sensory neurons contributing to the crushed nerve and an upregulation of PA-dependent activity in crushed nerve compared with sham counterparts during nerve regeneration. To further assess the role of the PA system during peripheral nerve regeneration, PA-dependent activity as well as recovery of sensory and motor function in the injured hindlimb were assessed in wild-type, tPA, uPA, and plasminogen knock-out mice. Protease activity visualized by gel zymography showed that after nerve crush, the upregulation of PA activity in the tPA and uPA knock-out mice was delayed compared with wild-type mice. Recovery of sensory function was assessed by toe pinch, footpad prick, and the toe-spreading reflex. All knock-out mice demonstrated a significant delay in hindlimb response to these sensory stimuli compared with wild-type mice. For each modality tested, the uPA knock-out mice were the most dramatically affected, showing the longest delay to initiate a response. These studies clearly showed that PAs were necessary for timely functional recovery by regenerating peripheral nerves.

Fig. 1.

Degeneration of sciatic nerve in wild-type mice 3 d after crush injury. A, Phase-contrast microscopy shows sciatic nerve degeneration 3 d after nerve crush.Dark-filled and dark-speckled circlesindicate collapsed axons. Degeneration of myelin sheaths appears as areas of dark myelin interspersed with clear areas of myelin. The degeneration also causes an increase in interaxonal space.B, Phase-contrast microscopy of sham nerve 3 d after surgery. Healthy axons appear as dark rings of myelin with clear centers. Nerve projections are uniformly spaced with no signs of axonal degeneration. Scale bar, 60 μm.

Fig. 2.

Gel zymography of PA-dependent activities in wild-type, tPA−/−, and uPA−/− mice 3 d after sciatic nerve crush. The PA proteolytic activity, as visualized by plasminogen-dependent casein zymography, shows a marked increase at the crush site compared with uncrushed sciatic nerve in both wild-type and knock-out mice (t, tPA; u, uPA).Lane 1, tPA standard, 0.8 mIU; lane 2, wild-type control nerve; lane 3, wild-type crushed nerve; lane 4, uPA−/− control nerve; lane 5, uPA−/− crushed nerve; lane 6, tPA−/− control nerve; lane 7, tPA−/− crushed nerve.

Fig. 3.

PA-dependent activity in crushed and control (sham + unoperated) sciatic nerve in wild-type and knock-out (tPA−/− and uPA−/−) mice. tPA (white bars) and uPA (gray bars) activities were assayed by gel zymography at each of the time points. Lytic zones on zymographs were analyzed using known amounts of recombinant tPA standard (0.1 IU rtPA = 375 densitometric volumes). Values are expressed as the volume of lysis (mean ± SEM; n ≥ 3). Crushed nerve activity for each PA was compared with sham nerve activity (at each time point), and wild-type PA-activity was compared with knock-out PA activity by t test analysis. A, In wild-type mice, uPA activity was elevated above sham at 3 hr, again at 1 d, and stayed elevated two- to threefold through 7 d after sciatic nerve crush. tPA activity increased above sham nearly threefold by 1 d and remained elevated through 7 d (*p ≤ 0.05). B, In tPA−/− and uPA−/− mice, uPA- and tPA-dependent activity in crushed nerve increased significantly above activity levels in sham nerve by 3 d after injury and remained elevated two- to threefold through 7 d (*p ≤ 0.05). There were no significant differences between PA-dependent activity levels of wild-type and knock-out mice. However, there was a 2 d delay compared with wild type in crush-induced tPA- and uPA-dependent activity in both the uPA−/− and tPA−/− knock-out mice, respectively.

Fig. 4.

Immunohistochemical analysis using GAP-43 antibody shows axonal regeneration of crushed sciatic nerve. Ten days after crush surgery, nerve sectioned distal to the crush site and sham nerve were immunostained with GAP-43 antibody as a marker for regeneration.A, Cross section of crush nerve ∼1.3 mm distal to the crush site shows extensive GAP-43 reactivity compared with sham (B). A, Inset, Longitudinal section of crushed nerve at area distal to crush also displays a high level of GAP-43 reactivity. B,Inset, Longitudinal section of sham nerve displays very low levels of GAP-43 reactivity. Scale bar, 10 μm.

Fig. 5.

Recovery of sensory and motor functions after sciatic nerve crush. Using sharpened forceps, a prick was given to the footpads of wild-type and knock-out (tPA−/−, uPA−/−, and plasminogen−/−) mice after sciatic nerve crush. The amount of time (in days) to elicit an initial response, as shown by a vocalization and foot withdrawal, was recorded. tPA−/− and uPA−/− mice were significantly delayed compared with wild-type mice on the footpad test. Plasminogen−/− mice were comparable with their wild-type counterparts. Time (in days) of the first indication of a return of the toe-spreading reflex was also noted by examining for lateral extension of the hindlimb accompanied by a foot flexure. Return of the toe-spreading reflex was significantly delayed for all knock-out mice compared with wild-type mice. For each test, uPA−/− mice were the most adversely affected (*p ≤ 0.05;n = 5).

Fig. 6.

Recovery of response to pinch in hindfoot digits after sciatic nerve crush. Wild-type mice and knock-out mice (tPA−/−, uPA−/−, and plasminogen−/−) received a pinch to the distal portion of each hindfoot digit using forceps. A vocalization and foot withdrawal were noted as a positive response. Time (in days) to elicit an initial response was recorded for each digit. Knock-out mice displayed an initial response to pinch that was significantly delayed compared with their wild-type counterparts for each digit (except digit2). uPA−/− mice demonstrated the longest delay. Digits are labeled 1 through 5, with1 the most medial and 5 the most lateral digit (*p ≤ 0.05; n = 5).

Fig. 7.

Total recovery of stimulus response in all digits after sciatic nerve crush in wild-type and knock-out (tPA−/−, uPA−/−, and plasminogen−/−) mice. A toe pinch was given to each hindfoot digit and time (in days) to elicit a response in all five digits was recorded. A positive response occurred when toe pinch elicited a vocalization and foot withdrawal. All knock-out mice showed significant delays for time taken to show a positive response in all digits compared with their wild-type counterparts. uPA−/− mice were the most severely impaired (*p ≤ 0.05 compared with wild type; n = 5).