Structural and functional recovery of electropermeabilized skeletal muscle in-vivo after treatment with surfactant poloxamer 188

Abstract

A critical requirement for cell survival after trauma is sealing of breaks in the cell membrane [M. Bier, S.M. Hammer, D.J. Canaday, R.C Lee, Kinetics of sealing for transient electropores in isolated mammalian skeletal muscle cells, Bioelectromagnetics 20 (1999) 194-201; R.C. Lee, D.C. Gaylor, D. Bhatt, D.A. Israel, Role of cell membrane rupture in the pathogenesis of electrical trauma, J. Surg. Res. 44 (1988) 709-719; R.C. Lee, J.F. Burke, E.G. Cravalho (Eds.), Electrical Trauma: The Pathophysiology, Manifestations, and Clinical Management, Cambridge University Press, 1992; B.I. Tropea, R.C. Lee, Thermal injury kinetics in electrical trauma, J. Biomech. Engr. 114 (1992) 241-250; F. Despa, D.P. Orgill, J. Newalder, R.C Lee, The relative thermal stability of tissue macromolecules and cellular structure in burn injury, Burns 31 (2005) 568-577; T.A. Block, J.N. Aarsvold, K.L. Matthews II, R.A. Mintzer, L.P. River, M. Capelli-Schellpfeffer, R.L. Wollman, S. Tripathi, C.T. Chen, R.C. Lee, The 1995 Lindberg Award. Nonthermally mediated muscle injury and necrosis in electrical trauma, J. Burn Care and Rehabil. 16 (1995) 581-588; K. Miyake, P.L. McNeil, Mechanical injury and repair of cells, Crit. Care Med. 31 (2003) S496-S501; R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524-4528; J.D. Marks, C.Y. Pan, T. Bushell, W. Cromie, R.C. Lee, Amphiphilic, tri-block copolymers provide potent membrane-targeted neuroprotection, FASEB J. 15 (2001) 1107-1109; B. Greenebaum, K. Blossfield, J. Hannig, C.S. Carrillo, M.A. Beckett, R.R. Weichselbaum, R.C. Lee, Poloxamer 188 prevents acute necrosis of adult skeletal muscle cells following high-dose irradiation, Burns 30 (2004) 539-547; G. Serbest, J. Horwitz, K. Barbee, The effect of poloxamer-188 on neuronal cell recovery from mechanical injury, J. Neurotrauma 22 (2005) 119-132]. The triblock copolymer surfactant Poloxamer 188 (P188) is known to increase the cell survival after membrane electroporation [R.C. Lee, L.P. River, F.S. Pan, R.L. Wollmann, Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo, Proc. Natl. Acad. Sci. 89 (1992) 4524-4528; Z. Ababneh, H. Beloeil, C.B. Berde, G. Gambarota, S.E. Maier, R.V. Mulkern, Biexponential parametrization of T2 and diffusion decay curves in a rat muscle edema model: Decay curve components and water compartments, Magn. Reson. Med. 54 (2005) 524-531]. Here, we use a rat hind-limb model of electroporation injury to determine if the intravenous administration of P188 improves the recovery of the muscle function. Rat hind-limbs received a sequence of either 0, 3, 6, 9, or 12 electrical current pulses (2 A, 4 ms duration, 10 s duty cycle). Magnetic resonance imaging (MRI) analysis, muscle water content and compound muscle action potential (CMAP) amplitudes were compared. Electroporation injury manifested edema formation and depression of the CMAP amplitudes. P188 (one bolus of 1 mg/ml of blood) was administrated 30 or 60 min after injury. Animals receiving P188 exhibited reduced tissue edema (p<0.05) and increased CMAP amplitudes (p<0.03). By comparison, treatment with 10 kDa neutral dextran, which produces similar serum osmotic effects as P188, had no effect on post-electroporation recovery. Noteworthy, the present results suggest that a single intravenous dose of P188 is effective to restore the structural integrity of damaged tissues with intact circulation.

Figure 1

Illustration depicting anesthetic and treatment injection sites (IP, JV respectively), hind limb electroporation path (dark areas of tail and left leg), and CMAP stimulation (Coil) and recording sites (CMAP) on anesthetized rat.

Figure 2

Representative phase contrast photomicrographs (40x) of H&E stained 7 μm sections of electrically shock biceps femoris muscles are shown. Electroporation damage is distributed very non-uniformly in each muscle reflecting the stochastic nature of electroporation. The more shocks the more severe and the more uniform the damage. Longitudinal sections from muscle exposed to (a) 3 shock pulses exhibit a low percentage of damaged cells with mostly normal cytoskeletal architecture and some interstitial edema; (b) muscles subjected to 6 shocks manifest damage to 20–30% of the cells with more edema and (c) muscle subjected to 12 shocks have marked "contraction-band necrosis" to most cells and severe edema. Biopsies were taken 6 hours after electrical shock.

Figure 2

Representative phase contrast photomicrographs (40x) of H&E stained 7 μm sections of electrically shock biceps femoris muscles are shown. Electroporation damage is distributed very non-uniformly in each muscle reflecting the stochastic nature of electroporation. The more shocks the more severe and the more uniform the damage. Longitudinal sections from muscle exposed to (a) 3 shock pulses exhibit a low percentage of damaged cells with mostly normal cytoskeletal architecture and some interstitial edema; (b) muscles subjected to 6 shocks manifest damage to 20–30% of the cells with more edema and (c) muscle subjected to 12 shocks have marked "contraction-band necrosis" to most cells and severe edema. Biopsies were taken 6 hours after electrical shock.

Figure 3

Plot characterizing the development of edema injury over time. A 5 x 5 mm ROI was selected, and mean T₂ values in a shocked leg (closed circles, ± SD, n = 25 pixels) and non-shocked leg (open circles, ± SD, n = 25 pixels) as a function of time after electroporation. In this case lactated ringers was used as treatment and injured tissue was followed over time

Figure 4

CMAP amplitudes in control and injured legs of rats receiving 9 and 12 shocks, respectively 12 shock (n=10), 12 shock control (n=8), 9 shock (n=17), 9 shock control (n=15). All animals received control LR treatment. At 5h, the sciatic nerve is blocked with lidocaine to verify that CMAP is nerve stimulated.

Figure 5

(a) Plot of T₂ values for three different muscle samples. (b) The characteristic hydration levels for muscle samples of interest. SOL – soleus, EDL – extensor digitorum longus, BFM – biceps femoris muscle. All animals received a 1 mL bolus treatment as described. For each treatment of all muscles, n = 5. Two sets of p-values were calculated to determine treatment significance. P188, between shocked LR and P188-treated muscle samples; Dextran, between shocked LR and Dextran-treated muscle samples. The first set is: p = 0.046 (SOL), p = 0.042 (EDL) and p = 0.952 (BFM). The second set of p- values is: p = 0.728 (SOL), p = 0788 (EDL) and p = 0.578 (BFM)

Figure 6

Average number of pixels for each T₂ value in the interval 30–100 ms for healthy (a) LR-treated (b) and P188-treated (c) rats; In (d), we can see the difference in the magnetic signal intensity distribution between P188-treated and healthy rats (bright) and between LR-treated rats and healthy rats (dark) suggesting the evolution of the volume of injury (n = 5).

Figure 6

Average number of pixels for each T₂ value in the interval 30–100 ms for healthy (a) LR-treated (b) and P188-treated (c) rats; In (d), we can see the difference in the magnetic signal intensity distribution between P188-treated and healthy rats (bright) and between LR-treated rats and healthy rats (dark) suggesting the evolution of the volume of injury (n = 5).

Figure 7

CMAP amplitudes in control, shocked (LR-treated), and shocked P188-treated rats. At 5h, the sciatic nerve is blocked with lidocaine to verify that CMAP is nerve stimulated. The unshocked contralateral control leg CMAP values were not significantly different between LR-treated and P188-treated groups, so control data was obtained from the LR-treated group as shown. Unshocked Control (n=8), P188 treated (n=3), LR treated (n=8). All animals received 9 shocks. All values are shown as mean +/− SEM. p = 0.0270. This is the two tailed p value calculated from an unpaired t-test at the 300 minute time point, comparing shocked P188 treated to shocked LR treated.

Figure 8

The greyscale contrast for characteristic T₁ and T₂ - weighted MRI images of control (left), injured (middle) and P188 treated (right) muscle samples suggesting the recovery of the water balance across the plasma membrane after the treatment with P188. One can observe that the grey level characterizing the magnetic signal of water in P188 treated muscle samples is approaching that corresponding to the control muscle samples. The parameters for recording T₁ - weighted MRI images were TR = 400 ms and TE = 5 ms. T₂ - weighted MRI images correspond to TR = 2s and TE = 60 ms.