Preservation of neuromuscular function in symptomatic SOD1-G93A mice by peripheral infusion of methylene blue

Experimental Neurology 285 (2016) 96–107

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Research Paper

Preservation of neuromuscular function in symptomatic SOD1-G93A mice by peripheral infusion of methylene blue☆ Janet D. Talbot a, John N. Barrett a,b, Doris Nonner a, Zhongsheng Zhang a, Kyle Wicomb a, Ellen F. Barrett a,b,⁎ a b

Department of Physiology and Biophysics, University of Miami Miller School of Medicine, P.O. Box 016430, Miami, FL 33101, USA Neuroscience Program, University of Miami Miller School of Medicine, Miami, FL, USA 33101

a r t i c l e

i n f o

Article history: Received 6 May 2016 Received in revised form 31 July 2016 Accepted 23 August 2016 Available online 25 August 2016 Keywords: Amyotrophic lateral sclerosis ALS Methylene blue Motor nerve terminal Muscle contraction Neuromuscular junction Skeletal muscle Superoxide dismutase 1 SOD1-G93A

a b s t r a c t In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3 weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400 μM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p b 0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the noninfused side (p b 0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1 μM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1G93A mice, even at late stages of disease. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Transgenic mouse models that express mutant forms of human superoxide dismutase 1 (SOD1) reproduce many aspects of the clinical disease of amyotrophic lateral sclerosis (ALS, Turner et al., 2013). In these mice some of the earliest morphological signs of motor neuron degeneration occur in the motor axons and motor nerve terminals that innervate fast muscle fibers (Fischer et al., 2004; Frey et al., 2000; Gould et al., 2006; Pun et al., 2006; Schaefer et al., 2005). These findings are consistent with the hypothesis, originally proposed by Fischer et al. (2004), that the motor neuron pathology in ALS includes a distal axonopathy, i.e., a dying-back pathology. Most research with mouse models of ALS Abbreviations: ALS, amyotrophic lateral sclerosis; BgTx, α-bungarotoxin; EDL, extensor digitorum longus muscle; ETC, electron transport chain; fALS, familial amyotrophic lateral sclerosis; FF, fast, fatiguable muscle; FUS, fused in sarcoma/ translated in liposarcoma; MB, methylene blue; SOD1, superoxide dismutase 1; TA, tibialis anterior muscle; TDP-43, transactive response DNA-binding protein 43; YFP, yellow fluorescent protein. ☆ None of the authors had a conflict of interest. ⁎ Corresponding author at: Department of Physiology & Biophysics R-43, University of Miami Miller School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136, USA. E-mail address: [email protected] (E.F. Barrett).

http://dx.doi.org/10.1016/j.expneurol.2016.08.013 0014-4886/© 2016 Elsevier Inc. All rights reserved.

has focused on the spinal cord, but multiple interventions found to preserve motor neuron cell bodies have failed to preserve their peripheral connections with muscle (reviewed by Dadon-Nachum et al., 2011; Fischer and Glass, 2007; Gould and Oppenheim, 2007; Murray et al., 2010; also Parone et al., 2013). Thus preservation of motor neuron somata, though necessary, is not always sufficient for preserving neuromuscular transmission and hence motor function. Some early peripheral damage occurs in mitochondria within motor terminals, and this mitochondrial dysfunction increases as the disease progresses (reviewed by Barrett et al., 2011). Thus we hypothesized that peripheral application of mitochondrial-protective drugs might prolong neuromuscular function in these mice. This initial proof-of-concept study used methylene blue (MB), which readily crosses cell membranes, accumulates within mitochondria, and can enhance respiration in both healthy and stressed mitochondria. MB has redox-cycling properties and has been demonstrated to increase O2 consumption, as well as synaptic transmission and aspects of memory, in multiple regions of the central nervous system (reviewed in Oz et al., 2011; Rojas et al., 2012; also Rodriguez et al., 2016). MB also protects brain regions exposed to a variety of stresses. For example, Tretter et al. (2014) demonstrated that MB increases O2 consumption and ATP production in brain mitochondria exposed to inhibitors of complex I or complex III (see also

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Lin et al., 2012). Additional reported beneficial effects of MB include reduced production of superoxide, and increased production of complex IV (= cytochrome c oxidase) and antioxidant defense enzymes. MB also modulates various ion channels, neurotransmitter systems and inflammatory mediators. In addition, for the present study, the blue color and fluorescence of MB's oxidized state facilitated analysis of drug distribution. MB was infused via an osmotic pump into the anterior muscle compartment in one hind limb of SOD1-G93A mice, beginning at a late presymptomatic age and ending as mice reached symptomatic, end-stage disease. Analysis focused on two vulnerable muscles in this compartment, tibialis anterior (TA) and extensor digitorum longus (EDL). In wild-type mice these muscles are composed mainly of fast, fatiguable (FF) muscle fibers (type IIB, Augusto et al., 2004; Hegedus et al., 2008), and in mutant SOD1 mice motor nerve terminals innervating limb FF muscles are the first to degenerate (Frey et al., 2000; Hegedus et al., 2007; Pun et al., 2006; Schaefer et al., 2005). Peripherally-initiated drug effects were assessed by pairwise comparisons of neuromuscular function and structure in MB-infused muscles vs. muscles in the contralateral non-infused limb of the same mouse. Comparison with the noninfused limb controlled for (1) effects mediated by any circulating MB, which would be available to muscles in both limbs, and (2) disease progression during the drug infusion interval. 2. Materials and methods Experiments used mice that expressed human SOD1-G93A (bred from founders purchased from Jackson Labs, Bar Harbor, ME; B6.CgTgN(SOD1-G93A)1Gur/J, stock #4435). To facilitate identification of motor terminals in measurements of endplate innervation, male SOD1-G93A mice were crossed with female mice (wild-type SOD1) that expressed yellow fluorescent protein (YFP) in many neurons (including motor neurons), but not in muscle or Schwann cells (founders from B6.Cg-Tg(Thy1-YFP)16Jrs/J, Jackson Labs, Bar Harbor ME, stock #3709). YFP expression facilitated morphological assessment of endplate innervation (see below). Tail clips obtained between postnatal days 18–20 were used to detect expression of YFP by fluorescence microscopy and expression of human SOD1 by PCR (performed in house as described in Vila et al., 2003, or by Transnetyx, Cordova, TN). Mice were housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All experiments were carried out using protocols approved by the University of Miami Animal Care and Use Committee. Mice were killed by an overdose of isoflurane anesthetic (3%) followed by cervical dislocation. 2.1. Osmotic pump implantation MB was infused via a 4 week osmotic mini-pump (model 1004, ALZET, Cupertino, CA) filled with a physiological saline that contained 0, 100, 200 or 400 μM MB, infused at ~ 0.11 μL/h. The pump was implanted subcutaneously between the shoulder blades and sutured to connective tissue. Polyethylene tubing (PE-60, Becton Dickinson & Co., Parsippany, NJ) exiting the pump was guided subcutaneously into the anterior compartment of one hind limb (diagram in Fig. 1A). The technique that most reliably achieved optimal placement of the drug infusion catheter between EDL and TA muscles (diagrammed in Fig. 1B) involved first positioning a suture thread, and then using this thread to guide the infusion tubing into place, as follows: A 2 mm length of 6-0 suture needle (attached to suture thread) was threaded between the two muscles in a distal-to-proximal direction (#1 → #2 in Fig. 1B). A magnetized needle (coupled to a powerful magnet) was then used to pull the suture needle vertically out of the proximal (“knee”) end of the anterior compartment (#2 → #3 in B). The suture thread thus traversed an L-shaped path through the anterior compartment. The infusion catheter from the osmotic pump was then attached to this suture thread and pulled into the anterior compartment along this L-shaped

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pathway in a proximal to distal direction (#3 → #2 → #1 in B). Successful execution of this technique allowed approximately equal drug exposure for both EDL and TA motor terminals with minimal muscle damage. In the section of tubing that coursed between EDL and TA muscles, the tubing was pulled down to a diameter of ~ 0.2–0.3 mm (using heat). The last 5 mm length was perforated with ~0.04 mm diameter holes spaced at ~0.5 mm intervals, to facilitate drug delivery throughout the anterior compartment. The distal end of the tubing was sealed using heat. Anesthesia for this sterile survival surgery was initiated using isoflurane (1.5%) and maintained at 0.7% isoflurane, delivered via a nose cone using a Matrx vaporizer and scrubber unit (Midmark, Orchard Park, NY). Anesthetized mice were placed on a heated pad to maintain body temperature. Skin openings were closed with fine (50) suture thread. Post-procedure pain control was provided by subcutaneous injection of buprenorphine (2 injections of 0.05–0.1 mg/kg on the first post-operative day). Mice with implanted mini-pumps were housed in individual cages and monitored daily by lab personnel and veterinary technicians. The osmotic pump was implanted at a late pre-symptomatic age, so that when drug infusion was complete 4 weeks later, mice would remain viable but exhibit obvious motor symptoms such as hindlimb weakness. At this symptomatic disease stage the majority of endplates in EDL and TA of the non-infused limb are denervated (see Figs. 5, 6A), facilitating detection of any beneficial effect of the drug on the infused side. Muscle structure and function were assayed when (a) the pump had been in place for 4 weeks and the mouse exhibited limb weakness, or (b) the mouse reached end-stage disease, defined as inability to right itself. In the rare situation that the mouse exhibited no motor symptoms 4 weeks after pump implantation, the osmotic pump was replaced with another pump containing the same drug concentration and the mouse was sacrificed when motor symptoms became evident. The 11 most extensively analyzed mice (400 μM MB mice included in Figs. 4–6) were exposed to infused drug for 20–47 days. Our mouse colony included some SOD1-G93A/YFP mice that developed obvious motor symptoms early (~ 100 days old) and others that developed motor symptoms ~ 80 days later and survived longer (210–240 days) than the founder SOD1-G93A mice. Pump implants were timed accordingly. The later onset suggests protective genes in the C57BL/6 background (Heiman-Patterson et al., 2011); quantitative PCR performed by Transnetyx indicated that the later onset was not due to a reduced copy number of the mutant human SOD1 gene. Four of the extensively analyzed mice were in the early-onset group (pump implanted at 88– 97 days); the remaining 7 were in the late-onset group (pump implanted at 174–194 days). The early- vs. late-onset groups were analyzed together because muscles in each infused limb had matched controls in the non-infused limb of that mouse. Criteria for inclusion in the analysis were (1) drug was infused for at least 20 days prior to development of end-stage disease and (2) dissection revealed proper placement of the infusion line between EDL and TA, without damage to the muscle tendons involved in recording muscle contractions (see below). 2.2. Measurement of muscle contractions in response to nerve and direct muscle stimulation Twitch and tetanic (0.5–0.6 s at 50 Hz) contractions evoked by indirect (motor nerve) or direct muscle stimulation were assayed in TA and EDL of both hind limbs, using methods similar to those described by Kalmar et al. (2008). Mice were anesthetized with 1–2% isoflurane, and the EDL and TA muscle tendons were dissected free at the distal end, leaving the proximal muscle insertion and nerve entry zones intact. The distal tendon was connected via a thread to a force transducer (F703C, Grass Instrument Co, Quincy, MA). The initial length of the muscle was adjusted to yield the maximal twitch tension in response to stimulation. Action potentials were evoked in the sciatic nerve by passing current between 2 platinum wires (31 gauge) embedded in a 5 mm

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Fig. 1. Placement of drug infusion line in hind limb anterior compartment. A, Mouse diagram indicates that the osmotic pump was inserted subcutaneously between the shoulder blades in the upper back; its outflow line was guided subcutaneously along the back and down into the anterior compartment of one hind limb. B, Pathway traversed by the catheter linking the outflow of the osmotic pump with TA and EDL muscles in the anterior compartment. The catheter region between these muscles was perforated to allow drug exit. Numbers 1–3 relate to the procedure used to implant the perfusion line, as described in the text. Drawings representing EDL, TA and catheter were superimposed on a photograph of mouse hindlimb muscles. C, MB staining (blue) in dissected TA and EDL muscles after fixation with paraformaldehyde.

long U-shaped stimulation cuff constructed from 3 mm diameter thinwalled silicon tubing. The amplitude of the brief (0.3 ms) depolarizing pulse (Grass S48 stimulator) was adjusted to about twice that needed to evoke a maximal peak tension (usually 1–4 V). Direct stimulation (5 V) was applied via a pair of platinum electrodes placed across the midsection of the muscle. The usual order of recording was: (1) indirect and direct twitch and tetanic stimulation of TA and EDL on the non-infused limb, (2) the same measurements on the infused limb, (3) repeat measurements on the non-infused limb to check for rundown. Peak contractile force was measured from digitized records (sample records in Fig. 2, Digidata 1440A A → D converter, pClamp 10.0 software, Axon Instruments/Molecular Devices, Sunnyvale, CA). In fALS mice (as in ALS patients, Ravits and La Spada, 2009) motor symptoms often exhibit spatial heterogeneity, such that weakness in one hind limb might appear well before weakness in the other hind limb becomes apparent. Thus as one control we compared muscle contractions in the two hind limbs of symptomatic SOD1-G93A mice that had no pump implanted (see no pump data in Fig. 3). Another control group consisted of SOD1-G93A mice implanted with pumps that contained only physiological saline (see 0 MB in Fig. 3). We also tested the effect of infusing MB into 3 mice expressing YFP only (no mutant SOD1). 2.3. Assessment of endplate innervation After muscle contractions were assayed, the mouse was killed (see above) and transcardially perfused, first with heparin saline (2 min),

then with 4% paraformaldehyde (in phosphate-buffered saline, 20 min). Muscles were dissected (complete with tendon-attached threads, noting and photographing the placement of the drug infusion catheter) and placed in 4% paraformaldehyde for 1–2 h on ice. EDL and TA were then further dissected (in saline) to facilitate confocal imaging. This dissection separated the 4 heads of the EDL and split the TA muscle longitudinally to achieve two thin sheets. Muscles were incubated with labeled α-bungarotoxin (BgTx, Alexa Fluor 594 conjugate, 25 μg/mL, Invitrogen, Carlsbad, CA) in physiological saline for 2 h on a vertical shaker. The BgTx was then washed out for 2 h, again with vertical shaking. Preparations were kept overnight at 4 °C, placed on the vertical shaker for an additional 2 h the following day, then mounted in Vectashield hard-set (Vector Laboratories, Burlingame, CA) and stored at 4 °C. Each muscle was imaged using a spinning disk confocal microscope to determine the percentage of BgTx-labeled endplates that were innervated by YFP-labeled motor terminals, as described in Talbot et al. (2012). Muscle regions containing BgTx-labeled endplates were identified, and z-stacks of images were collected with the bottom image of each stack set to the lowest edge of the muscle and the top image set to include the uppermost labeled endplate. Analysis used a custom macro written in Image J, whereby each level in the stack was represented by 3 interleaved fluorescent images, one for the endplate marker BgTx, one for the nerve marker YFP, and one with the merged, pseudocolored image. BgTx-labeled endplates were imaged using excitation with a 568 nm diode laser (Melles-Griot, Albuquerque, NM) and long pass emission filter (N590 nm). YFP-labeled nerve was imaged

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Fig. 2. Comparison of twitch (left trace) and tetanic (right trace) recordings of contractile force in non-infused (upper) and MB-infused (lower) TA muscles of an SOD1-G93A mouse. The anterior compartment was infused for 27 days from an osmotic pump containing 400 μM MB. Responses were elicited by a single (twitch) or a train (50 Hz tetanus) of supra-threshold stimuli delivered to the sciatic nerve. Both twitch and tetanic forces recorded from the MB-infused TA were larger than those recorded from the contralateral non-infused TA.

using excitation with an argon 488 nm laser (Laser Physics, West Jordan, UT) with 535 ± 20 nm bandpass emission filter. Four non-overlapping stacks of images were collected for each of the 4 EDL heads and 16 stacks for each TA (20× Nikon objective, NA 0.75). Each stack contained 30–190 levels, and at least 200 endplates were sampled for each muscle. Z-projections of representative stacks are illustrated in Fig. 5. To determine the percentage of innervated endplates, an observer blinded to the experimental treatment analyzed single optical sections, using a slider to ascend from the bottom to the top of the image stack. Postsynaptic endplate regions were identified and marked on the BgTx image. The observer then examined the corresponding YFP and merged images to determine whether or not there was a presynaptic terminal at the same position both in depth (z-axis) and in the xy plane. An endplate was considered innervated if the YFP-filled motor terminal remained connected to its pre-terminal axon and occupied at least 50% of the BgTx-labeled endplate. The YFP-labeled image stacks described above were also analyzed to count the number of intact vs. degenerating axons, also by an observer blind to the experimental treatment. Degenerating axons were identified as disconnected YFP-labeled fragments arranged in a line or curve (see arrows in Fig. 6C), based on observations in Beirowski et al. (2004). The number of intact and fragmented axons in each z-stack field was counted. This assay would miss axons that had degenerated more than ~10 days previously, which would have been removed by phagocytosis. Some axons were broken as fixed TA muscles were split (see above), but in this case the broken axons looked intact up to the break, very different from the pattern associated with degenerating axons. 2.4. Estimation of methylene blue concentration in infused muscle MB exists in two forms, a reduced (leuko) form that is colorless and an oxidized, fluorescent, positively-charged blue form. MB's blue color

was not evident when MB-infused muscles were first dissected, but emerged within a few minutes following tissue fixation with paraformaldehyde (Fig. 1C). This observation suggests that under physiological conditions (including the lower-than-atmospheric oxygen pressure in vivo) MB exists mainly in its colorless leukoMBH2 form, which upon fixation (and exposure to air) converts to the colored oxidized form. Muscle cells contribute to reduction of MB, because addition of dissected muscles to a 2 mL sealed tube containing MB (blue form) turned the solution colorless; the blue color reappeared when the muscles were subsequently fixed. This blue staining in fixed tissue was not permanent; MB slowly diffused out of the muscles during overnight storage. No MB fluorescence was detected in freshly fixed sciatic nerve N5 mm from the infused anterior compartment, in lumbar spinal cord, or in muscles from the non-infused limb. These findings suggest that the circulating concentration of MB was very low, and either that MB was not retrogradely transported, or that any retrogradely transported MB was metabolized. Thus the beneficial effects of MB reported here likely originated within the infused muscle compartment. In the infused anterior compartment the concentration of MB was, as expected, more intense near the former location of the infusion tube, becoming fainter near the periphery (Fig. 1C, ~50% decay in 1 mm). Dissection disclosed that a connective tissue sheath formed around the infusion line; this sheath likely made the concentration of the infused drug more uniform along the tube, reducing the effect of “hot spots” near the holes in the infusion line. With 400 μM MB in the pump there was no evidence of toxicity in tissues nearest the infusion line, but toxicity was evident in a few experiments in which pump [MB] was increased to 2.5–5 mM. Approaches used to estimate the MB concentration in the infused anterior muscle compartment are detailed in Appendix A. These measurements and calculations suggest that with 400 μM MB in the infusion pump, the estimated total extracellular concentration within the

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parametric statistical tests were applied, such as the Mann-Whitney signed rank test. Other statistical tests are specified in the text. The dose-response curve in Fig. 3 was constructed using the sum of the EDL and TA force values measured in a given limb; the bar graph in Fig. 6C also summed EDL and TA values. Bar graphs in Figs. 4 and 6A analyzed EDL and TA muscles separately. 3. Results 3.1. MB infusion increases contractile force in response to nerve stimulation in mutant SOD1 mice

Fig. 3. Effect of [MB] in the infusion pump on the % change in peak contractile forces evoked by nerve stimulation in SOD1-G93A mice. Each point represents the (infused minus non-infused) difference for an individual mouse (X = twitch; circle = 50 Hz tetanus). Squares connected by dashed lines indicate the mean tetanic value for the indicated [MB]. The percentage increase for twitch force was calculated as follows: (((EDL twitch + TA twitch on infused side) − (EDL twitch + TA twitch on non-infused side)) / (average non-infused EDL twitch + TA twitch for all mice at that MB concentration)) × 100. Percentages for tetanic force were calculated using a similar equation. The correlation between [MB] and % force increase on the infused side was significant (p b 0.01 for range from 0 to 400 μM MB, Pearson correlation analysis). The Mann-Whitney signed rank test indicated that for 400 μM MB the differences between forces measured on the MB-infused and non-infused sides were very unlikely to be explained by random variation (p b 0.01, see Fig. 4). In addition the Mann-Whitney U test indicated a significant difference between the % change values measured for 400 μM MB vs. saline-only infusions (p b 0.01). Points to the left of the ordinate represent mice with no implanted pump that were analyzed at end-stage disease; for these mice we arbitrarily subtracted left limb from right limb values. N = 11 mice for 400 μM MB, 6 for 200 μM, 9 for 100 μM, 7 for saline-only, and 10 for No pump.

anterior compartment was ~1–2 μM, with total intramuscular concentrations about 5× greater. 2.5. Reagents MB was from Sigma/Aldrich (M9140, St. Louis, MO). Other reagent sources: isoflurane, Piramal Healthcare Ltd. (Andhra Pradesh); Alexa Fluor 594 conjugate of α-bungarotoxin, Invitrogen (Carlsbad, CA); all other reagents (Sigma-Aldrich). 2.6. Statistical tests Statistical tests were performed using GraphPad Prism and Instat (GraphPad Software, La Jolla, CA). Data for most of the tested groups satisfied the test for normality, justifying use of paired t-tests to compare muscles in infused vs. non-infused hind limbs. In some cases non-

Fig. 2 shows sample measurements of twitch and tetanic forces from TA in response to indirect (nerve) stimulation in an SOD1-G93A mouse whose osmotic pump contained 400 μM MB. Comparison of records from the non-infused (upper) and infused (lower) muscles indicates an increase in both twitch and tetanic force on the infused side. Dose-response data in Fig. 3 indicate how the % change in peak contractile force varied with the concentration of MB in the osmotic pump. These measurements summed together the forces measured for fast muscles EDL and TA in the anterior compartment. The % change in summed peak contractile force (plotted on the ordinate) was calculated as the difference between the peak force measured in the MB-infused limb minus the peak force measured in the contralateral non-infused limb of that mouse. This difference was normalized by dividing by the average value for the non-infused limb measured in all mice exposed to that MB concentration. Data from an individual mouse are indicated by two symbols, X for twitch and circle for tetanus. An average (infused minus non-infused) force exceeding zero suggests a beneficial effect of localized peripheral infusion. Squares connected by a dashed line in Fig. 3 indicate the average % difference in tetanic force at each tested MB concentration (0, 100, 200, 400 μM MB in the pump). Data for mice infused only with physiological saline (0 MB) demonstrate that infusion alone had no significant beneficial effect on contractile force. There was a strong correlation between increasing [MB] and increased force on the infused side (dashed line, p b 0.01 for range from 0 to 400 μM MB, Prism Pearson correlation analysis). The average increase in force for 400 μM MB was ~100% (additional statistical tests are summarized in Fig. 3 legend). This beneficial effect was due to the localized peripheral infusion of MB, because if MB's effects had instead been exerted mainly by the small circulating concentration of MB (which should affect both sides equally), then one would expect (on average) no significant difference between the contractile forces measured in infused vs. non-infused muscles. There was no significant difference between values measured for saline-only mice and mice with no implanted pump (“No pump” in Fig. 3). In both cases the average inter-limb difference was not significantly different from zero. These results thus indicate that localized peripheral infusion of a sufficient concentration of MB had a beneficial effect on neuromuscular function that was evident even in late-stage symptomatic SOD1-G93A mice. Based on these findings, the results presented in the following figures were all obtained with the most effective concentration, 400 μM MB, in the osmotic pump. Fig. 4A,B plot the amplitude of the peak twitch contractions for EDL (A) and TA (B) measured in the 11 mice treated with 400 μM MB. Values for non-infused and infused limbs for each individual mouse are connected by a line. Fig. 4C,D show similar plots for the peak tetanic contractions measured in these same mice. Contractile force was almost always greater for muscles in the MB-infused limb. Force values for TA usually exceeded those for EDL because TA is a larger muscle. Bar graphs in Fig. 4E show that, on average, force values on the infused side were about 2-fold greater that those on the non-infused side. Thus MB infusion initiated in late pre-symptomatic mice was able to preserve (at least partially) neuromuscular function in both these fast muscles even at end-stage disease.

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Fig. 4. Infusion of 400 μM MB increases EDL and TA muscle contractions evoked by both nerve (A–E) and direct muscle (F) stimulation. A and B, peak twitch contraction evoked in EDL and TA muscles in non-infused and MB-infused limbs. Lines connect values recorded in muscles from the same mouse. C and D, peak tetanic contraction evoked by 50 Hz stimulation in the same muscles studied in A,B. A positive slope indicates increased contraction force in the MB-infused limb. Note the different ordinates in A–D. E, Averaged amplitudes (±SEM) for the data in A–D. F, averaged amplitudes for similar records obtained using direct muscle stimulation in the same mice. The average MB-associated percentage increase with direct muscle stimulation (F) tended to be less than that for nerve-evoked muscle contractions (E). For nerve-evoked contractions (E) the difference between values from MB-infused vs. noninfused limbs was significantly different from zero at p b 0.01 or better for all groups tested (EDL twitch, EDL tetanus, TA twitch, TA tetanus; paired t-test). For the contractions evoked by direct muscle stimulation (F) the difference between values for infused vs. non-infused limbs was significantly different from zero at p b 0.02 or better for all 4 tested groups. The duration of MB infusion ranged from 20 to 47 days; n = 11 mice. mN = milliNewtons.

One might expect that in mice with a large MB-associated increase in contractile force a corresponding asymmetry between EDL- and TA-associated limb movements would have been evident. We did observe and document hind limb movements in late/end-stage mice, paying special attention to toe spread when the mouse was held inverted by the tail (an EDL-associated response) and to placement of the hind limb under the body (a TA-associated response). In some mice toe spread and limb placement were clearly better on the side whose EDL and TA muscles subsequently evidenced increased contractile force. However, other mice showed marked rigidity of both hind limbs, associated perhaps with dysfunction of other neurons in the spinal cord or brain (e.g. inhibitory interneurons, Dentel et al., 2013).

forces were measured in the MB-infused limb. This result indicates that some of the beneficial effects of localized MB infusion were exerted on the muscle itself, either directly or indirectly. One example of an indirect effect would be improved muscle strength attributable to MB-induced maintenance of endplate innervation (see below). The peak force in response to direct muscle stimulation (Fig. 4E) was on average greater than the response to indirect (nerve) stimulation (Fig. 4F), as would be expected from the significant number of denervated endplates present in fast limb muscles of symptomatic mutant SOD1 mice (e.g., Schaefer et al., 2005; Nguyen et al., 2012; also see Fig. 5).

3.2. MB infusion increases contractile force in response to direct muscle stimulation

To test whether preservation of endplate innervation also contributed to the increased contractile force measured in MB-infused muscles, we compared the percentage of endplates innervated by a motor terminal in muscles from infused and non-infused limbs. Fig. 5 shows representative pseudocolor fluorescence micrographs of projections of zstacks from the MB-infused (left column) and non-infused (right column) EDLs of one mouse. The YFP-expressing nerves appear green, and the α-BgTx-labeled endplates appear red. In the merged images (bottom row) the innervated endplates appear yellow (superposition of green and red), whereas denervated endplates appear red. Both MB-infused and non-infused EDLs showed evidence of denervated endplates in this end-stage mouse, but the percentage of denervated

The increased contractile force associated with peripheral MB infusion could have multiple possible mechanisms, including a beneficial effect on the muscles themselves and/or a beneficial effect on motor nerves/motor nerve terminals. To test for a beneficial effect on muscle, we measured twitch and tetanic contractions evoked by direct muscle stimulation in the infused and non-infused EDL and TA muscles of these same mice, using techniques similar to those described for nerve-evoked contractions in Figs. 3 and 4A–D. Fig. 4F shows the averaged results for direct muscle stimulation. In all cases higher contractile

3.3. MB infusion increases the percentage of innervated endplates

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Fig. 5. Sample fluorescence images from MB-infused (left) and non-infused (right) EDLs indicate a greater percentage of innervated endplates on the infused side. Muscle endplates (upper row, labeled with Alexa 594-α-BgTx) appeared similar in both muscles, but nerve processes (middle row, labeled with transgenically-expressed YFP) were more abundant on the MBinfused side. In the merged image (lower row) endplates occupied by a motor terminal (yellow) were more prevalent on the infused side. Each illustrated panel represents a zprojection of a stack of 90–120 confocal images, and thus includes endplates at different depths and orientations through the thickness of the muscle. Endplate innervation was assessed by analyzing images at each z-level of the image stack, as described in Materials and Methods. Calibration: 100 μm.

endplates was lower in the MB-infused EDL. Innervation within any one muscle had a heterogeneous distribution, especially on the non-infused side: some image stacks showed no innervated endplates, whereas others showed moderate or almost complete innervation. Such heterogeneity is perhaps to be expected late in the disease when only a few motor axons remain in fast muscles. Fig. 6A presents averaged data showing that MB infusion increased the percentage of innervated endplates in end-stage mice. Most endplates in non-infused muscles were denervated, with only 35% innervated in EDL and 30% in TA. MB infusion increased the innervated percentage to 65% in EDL and 43% in TA. A possible reason for the smaller effect in TA is that it is a thicker muscle than EDL; TA muscle layers more distant from the infusion line probably experienced a lower MB concentration than the more proximal layers. If MB's effects on nerve-evoked contractile force illustrated in Figs. 3-4 were due (at least in part) to an increase in endplate innervation, then one would expect a positive correlation between these functional and morphological measurements. Fig. 6B plots force vs. % innervated endplates for EDL in all analyzed mutant SOD1 mice treated with 400 μM MB (triangles for twitch, squares for tetanus). Filled symbols represent non-infused EDLs and open symbols represent MB-infused EDLs. Best fitting lines for twitch and tetanus are shown, indicating a strong positive correlation between force and endplate innervation (p b 0.001 for both twitch and tetanus, Pearson correlation analysis). In a similar plot for TA (not shown), the best-fitting lines also had positive slopes, but the correlation did not reach significance, probably due to reduced MB access to the more distant TA muscle layers.

Another prediction is that muscles with more functional innervation should show less discrepancy between contractions elicited by nerve (indirect) stimulation vs. muscle (direct) stimulation than muscles with less functional innervation. Consistent with this prediction and the morphological data of Figs. 5 and 6A, comparison of the nerve stimulation data of Fig. 4E with the direct muscle stimulation data of Fig. 4F indicates that the mean indirect/direct ratio for tetanic stimulation increased from 64% for untreated TA to 85% for MB-treated TA (p b 0.05), and from 57% for untreated EDL to 91% for MB-treated EDL (p b 0.001, unpaired two-tailed t-test). Axonal sprouts were evident on both MB-infused and noninfused sides. The number of fragmented (degenerating) and intact YFP-labeled axons/sprouts were counted in the same stacks of YFP images used to assess endplate innervation (see Materials and Methods). Fig. 6C shows a sample field containing both intact and fragmented axons; the graph shows that the percentage of fragmented (EDL + TA) axons was smaller on the MB-infused side. This result suggests that MB's beneficial effects extended to motor axons as well as motor terminals. The increase in endplate innervation in MBinfused muscles could be due to preservation of existing axons/sprouts and/or to enhancement of axonal sprouting from surviving motor axons to innervate endplates vacated by degenerated motor axons. Studies to distinguish between enhanced preservation vs. enhanced regeneration would benefit from use of a thinner muscle, (preferably one that could be imaged repeatedly during the course of MB treatment) and deconvolution software to make it easier to follow individual sprouts.

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Fig. 6. Effects of MB infusion on the percentage of endplates innervated by a motor nerve terminal (A) and the percentage of fragmented axons (C), and the relationship between muscle contractile force and endplate innervation (B). A, Average percentage of innervated endplates in MB-infused and non-infused EDL and TA (p b 0.01 for EDL, p b 0.05 for TA, paired t-test). B, Peak contractile force (mN, triangles = twitch, squares = tetanus) as a function of percent endplate innervation for EDL. Open symbols = MB-infused; closed symbols = non-infused. Lines (forced through 0,0) indicate best fit for twitch (r2 = 0.58) and tetanus (r2 = 0.61); the slopes of both lines were significantly different from zero (p b 0.0001, F-test). C, Fluorescence image of YFP-labeled axons in a non-infused EDL; arrows indicate fragmented axons. Calibration: 100 μm. Graph compares percentage of fragmented axons in TA + EDL muscles on non-infused vs. MB-infused sides (p b 0.01, paired t-test).

3.4. MB infusion does not improve neuromuscular function or structure in mice expressing wild-type SOD1 Contractile force and endplate innervation in anterior compartment muscles on infused and non-infused sides were also compared in 3 YFPexpressing mice (with no mutant SOD1) implanted with pumps

containing 400 μM MB. The ratio of infused/non-infused peak twitch force, calculated as in Fig. 3, averaged 86% in response to nerve stimulation, and 82% in response to direct muscle stimulation. Endplate innervation was 100% on both infused and non-infused sides. The reduced average force on the infused side was likely due to mechanical damage caused by the infusion line, because there was no denervation and the

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degree of force reduction was similar for both nerve and direct stimulation. A similar tendency toward lower contractile force was also apparent in mutant SOD1 mice infused with saline only (0 MB, Fig. 3). Although the sample size was small, these results suggest that MB infusion does not enhance neuromuscular function in mice that express only wild-type mouse SOD1. This finding is consistent with Tretter et al.'s (2014) finding that MB enhanced O2 consumption and ATP production in brain mitochondria stressed by exposure to inhibitors of complex I or III, but had no such effect in non-stressed mitochondria. However, with the measurements made in our study it would have been difficult to detect any beneficial effect of MB in wild-type preparations, where all endplates are innervated and neuromuscular transmission is always suprathreshold. 4. Discussion 4.1. Protection of motor terminal structure and function in late-stage disease The experimental design used in this proof-of-concept study, namely unilateral drug infusion into a muscle compartment, enabled pairwise comparisons between treated and untreated muscles in the same mouse. These comparisons demonstrated that peripherally- infused MB can protect neuromuscular structure and neuromuscular transmission in predominantly-fast limb muscles of SOD1-G93A mice, even at symptomatic stages of disease. The preservation of endplate innervation, combined with the beneficial effect on both nerve- and musclestimulated contractile force, indicate that distal motor axons, nervemuscle connections, and muscle strength were all protected. These beneficial effects of MB infusion likely had a peripheral origin, since we found no evidence for retrograde transport of MB into the spinal cord. MB effects may have been exerted directly on the infused peripheral structures (muscle, motor axons/terminals, Schwann cells). Alternatively or in addition, peripheral actions of MB may have exerted a protective effect via modification of retrogradely transported substances. We could not determine whether or not peripheral MB infusion increased survival of motor neuron somata in the present study because only a small percentage of motor neurons in the lumbar cord originated from the MB-infused anterior compartment, and we were not able to identify this small population in spinal cord sections (YFP expressed under the Thy1 promoter labels many spinal cord neurons in addition to motor neurons.) Answering this important question would require increasing the number of limb muscles exposed to MB, and bilateral retrograde labelling of the relevant motor neuron populations. 4.2. Sources of variability Although on average the contractile responses to both indirect (nerve) and direct (muscle) stimulation were larger in the MB-infused limb, there was considerable variability among mice, both in the values measured in the non-infused limb and in the percentage increase measured in the MB-infused limb. Some of this variability results from the heterogeneous expression of the disease itself, since in mutant SOD1 mice (as in ALS patients) disease processes become manifest at varying ages, and one limb may be affected earlier than the other. The infusion line was placed before any paralysis was obvious. If by chance disease expression happened to develop earlier in the infused limb, the percentage enhancement produced by MB would be less than if disease expression happened to be more severe in the non-infused limb. Another factor that probably affected the magnitude of the MB-induced increase was the position of the infusion tube relative to the endplate regions of these muscles. We attempted to place the infusion tube between EDL and TA such that MB would have access to the endplate regions of both muscles, but in some cases inspection at the time the muscles were dissected indicated that the tube was closer to

one muscle's endplate region than to the other. In such cases the muscle closer to the infusion tube usually exhibited the greater increase in force. Summing the forces for TA and EDL (as in Fig. 3) helped minimize the effect of this possible source of variability. 4.3. Possible mechanisms underlying MB's stress-protective effects The extracellular MB concentration that preserved neuromuscular structure and function in the infused muscle compartment was estimated as 1–2 μM, within the range of concentrations reported to be stressprotective in tissue culture studies (Daudt et al., 2012; Poteet et al., 2012; Xie et al., 2013; Yamashita et al., 2009) and brain mitochondria (Tretter et al., 2014). MB's ability to preserve neuromuscular function in late-stage disease could involve multiple mechanisms, including acting as a recyclable anti-oxidant, a potentiator of mitochondrial electron transport chain (ETC) activity, and an inhibitor of abnormal protein aggregation (reviewed in Dibaj et al., 2012; Oz et al., 2011). These actions would be expected to ameliorate the disruption of motor function that occurs in mutant SOD1 models of fALS (reviewed by Barber and Shaw, 2010; Barrett et al., 2011; Cozzolino and Carri, 2012; Kawamata and Manfredi, 2010). Fischer et al. (2012) present evidence that motor nerve terminals are especially vulnerable to oxidative injury. In fALS mice with compromised motor terminals MB might enhance neuromuscular transmission by increasing transmitter release (as reported at some central synapses, Rojas et al., 2012) and/or by inhibiting transmitter degradation by acetylcholinesterase (Pfaffendorf et al., 1997). MB's ability to function as a recyclable anti-oxidant (the basis for MB's clinical use to treat methemoglobinemia) allows it to serve as an alternative electron transporter, able to accept electrons from NADH at complex I of the respiratory chain (thereby reducing MB to leukomethylene blue, MBH2), and shuttle those electrons to cytochrome c and oxygen, thereby bypassing complexes I-III of the ETC (reviewed in Oz et al., 2011; Rojas et al., 2012). This ability would enhance the function of mitochondria in which these complexes were reduced or inhibited, and also reduce intramitochondrial formation of oxygen free radicals (O− 2 , Atamna et al., 2008; Furian et al., 2007; Rojas et al., 2009; Zhang et al., 2006). MB's ability to serve as an artificial electron acceptor could also divert electron flow away from the active sites of other oxidases where molecular oxygen is converted to superoxide (O− 2 , Oz et al., 2011). Protection of mitochondrial function may be especially critical for preserving motor terminal function. Mitochondria in motor nerve terminals temporarily sequester large stimulation-induced Ca2+ loads, a function that might be especially vital in motor terminals innervating fast, fatiguable muscles, which can discharge in high frequency bursts (Burke, 2004). Healthy mitochondria accelerate ETC activity in response to the depolarization associated with Ca2+ influx into the matrix (e.g., Talbot et al., 2007; reviewed in Cozzolino and Carri, 2012). Mitochondria in motor terminals of mutant SOD1 mice appear to lose this ability to accelerate respiration (reviewed in Barrett et al., 2011), consistent with Carrasco et al.’s (2012) finding of increased reliance on glycolysis in motor terminals of SOD1-G93A mice. Mitochondrial dysfunction would disrupt both the ATP-generating and Ca2+-regulatory functions of these organelles, thereby decreasing transmitter release during sustained activity. MB might also protect motor nerves and muscle by reducing production of mutant SOD1 and/or by decreasing mitochondrial accumulation of mutant SOD1. Abnormal oxidation can contribute to production of denatured SOD1 (e.g. Karch et al., 2009; Martins and English, 2014), so MB-induced reduction of intramitochondrial ROS production might benefit both motor axons and muscle by reducing the formation of denatured and/or mutant proteins (e.g. in rat SOD1-G93A motor axons, Sotelo-Silveira et al., 2009). Mutant SOD1 forms aggregates in mitochondria (e.g. Vijayvergiya et al., 2005), and reduction of these mitochondrial aggregates (with glutaredoxin 2) protected neuronal cells in vitro (Ferri et al., 2010). In addition, prevention of Ca2+ dysregulation

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protected against formation of protein aggregates in cultured motor neurons transfected to express human SOD1-G93A (Tradewell et al., 2011). MB is reported to reduce aggregation of TDP-43 (Yamashita et al., 2009), and to reduce stress imposed by accumulation of abnormal proteins in models of other neurodegenerative diseases (Alzheimer's: Ladiwala et al., 2011; Medina et al., 2011; Necula et al., 2007; Parkinson's: Sontag et al., 2012). Accumulation of denatured and/or misfolded proteins can produce endoplasmic reticular (ER) stress, and in worm and zebrafish models MB protected against TDP-43 toxicity by reducing ER stress (Vaccaro et al. (2013). Interestingly, intramuscular injections of salubrinal, which also protects against ER stress, preserved neuromuscular junctions and motor neurons in mutant SOD1 mice (Saxena et al., 2009). Note, however, that protective effects of MB are not limited to diseases characterized by abnormal protein aggregates (e.g., Legault et al., 2011). Further studies are needed to determine which (if any) of these mechanisms is most relevant for MB's protective effects on neuromuscular function and structure, and the time course of these effects (e.g., acute/sustained vs. chronic/cumulative). 4.4. Comparison with effects of systemic administration of MB in fALS models Reported effects of in vivo administration of MB in mutant SOD1 mice are mixed: Audet et al. (2012) reported no beneficial effect of MB in mutant SOD1 mice, but Lougheed and Turnbull (2011) and Dibaj et al. (2012) reported that systemic administration (oral, intraperitoneal) of MB delayed motor neuron death, motor dysfunction and disease onset. In their studies MB did not increase life span, but MB-treated mice spent more time in early-stage disease and less time in more severe, end-stage disease. Similarly, in worm and zebrafish mutant TDP43 and FUS models of ALS, MB protected motor neurons at early disease stages, but did not increase overall survival (Vaccaro et al., 2012). These results are consistent with the idea that MB ameliorates some disease process(es) common to multiple fALS models. In view of the reported benefits of systemic MB on motor neuron somata and our finding of beneficial results on neuromuscular function even at late stages of disease, we wondered why systemically-administered MB did not prolong survival in fALS animal models. Some possible reasons include: (1) Perhaps the circulating MB concentrations achieved in published systemic administration studies (these concentrations were not reported) were not adequate to produce the beneficial neuromuscular effects, due to an inadequate drug dose and/or to the fact that drug exposure was pulsatile rather than steady. (2) In our study most of the MB that accumulated within muscles of the infused hind limb compartment was converted to and remained in the reduced, colorless, uncharged leukoMBH2 form. Perhaps in late stage disease the increasing oxidative stress within the spinal cord makes it less able to convert systemically-administered MB to its reduced form, even though muscle cells retain this ability. Excessive accumulation of the positivelycharged, oxidized form of MB within negatively-charged compartments (e.g., mitochondrial matrix) might be detrimental. (3) Perhaps MB's beneficial effects were countered by other detrimental side effects. In Tretter et al.’s (2014) study of brain mitochondria the MB-induced increase in O2 consumption was always accompanied by increased production of hydrogen peroxide (H2O2); this problem might be overcome by administering MB along with a synthetic catalase mimetic. Other problems might arise from MB's preferential accumulation within certain cells (reviewed by Oz et al., 2011). 4.5. Possible clinical implications In most ALS patients the disease is diagnosed after the onset of motor weakness, suggesting that some muscle endplates have already become denervated. Thus one goal of effective therapeutics is to preserve the remaining motor terminals. Support of these remaining

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peripheral connections may be especially challenging at late disease stages, when the remaining motor neurons are under the extra metabolic stresses associated with reactive microglia, axonal sprouting and attempted re-innervation of muscle endplates denervated by the death of other motor neurons. Nguyen et al. (2012) demonstrated that in symptomatic SOD1-G85R mice almost all of the motor terminals that remained in a fast limb muscle retained some ability to respond to motor nerve stimulation. Since in the present study the infusion of MB began at a late pre-symptomatic stage and lasted until mice reached end-stage disease, our results demonstrate that the structure and function of these remaining terminals can be prolonged via mechanisms initiated peripherally. The unilateral drug infusion technique described here is labor-intensive and thus not suitable for high-throughput studies aimed at rapid screening of candidate ALS therapeutic drugs. But techniques such as ours aimed at testing ability to preserve mature functional neuromuscular junctions are crucial at later stages of drug testing, since the literature contains numerous examples of drugs that preserved motor neuron somata but failed to preserve their peripheral connections with muscle (see Introduction). 5. Conclusions In summary, this paper describes a novel technique to identify drugs that preserve neuromuscular function in a mouse model of familial ALS. We show that a localized, unilateral, ≥ 3 week infusion of methylene blue (MB) into a hind limb muscle compartment increased both the percentage of innervated endplates and the strength of twitch and tetanic contractions in fast muscles of late-stage SOD1-G93A mice. Contractions evoked by both nerve (indirect) and muscle (direct) stimulation were increased. Results suggest that these beneficial effects were initiated peripherally, and involved the reduced form of MB, with an estimated average extracellular [MB] of ~1–2 μM in the infused muscle compartment, and a ~5-fold greater concentration within muscle fibers. Treatments that act peripherally to preserve neuromuscular function might complement treatments that preserve motor neuron somata, creating an effective combination therapy to prolong function and possibly survival in ALS patients. Funding This study was supported by the Muscular Dystrophy Association (#186832) and by funds provided by the Scientific Advisory Committee of the University of Miami Miller School of Medicine. These funding sources had no influence on study design, data analysis, or the writing and submission of this report. Acknowledgements We thank Dr. Gavriel David for valuable discussions and for loaning the equipment used for anesthesia and for measurements of muscle contraction. This work is dedicated to the memory of JB's graduate mentor, Dr. Wayne Crill, who succumbed to an ALS-like disease. Appendix A A.1. Estimating MB concentrations in the infused muscle compartment One approach used EDL and TA muscles that had been infused for several weeks from an osmotic pump containing 400 μM MB, as described in Materials and Methods. Muscles were dissected, fixed and weighed. MB was extracted and converted to its oxidized colored form using acetonitrile (100%, 200 μL for 5–20 mg of muscle tissue, O'Leary et al., 2010). MB fluorescence was measured using a platereader (Victor 1420, Wallac/Perkin Elmer, Akron, OH), yielding an average total [MB] ranging from 2 to 8 × 10−6 μM/mg tissue, or ~2–8 μM.

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Another approach calculated estimates of the steady-state concentration of MB from the infusion rate (0.11 μL/h × 400 μmol/L), the volume of the anterior compartment, and the rate of MB disappearance from this compartment. The volume of the anterior compartment (which includes muscles in addition to EDL and TA), calculated from wet weight, was ~ 60 μL for end-stage mutant SOD1 mice (compared to ~96 μL for age-matched wild-type mice). The rate of MB disappearance from this compartment was calculated by injecting a known amount of MB into the anterior compartment with a syringe (20 μL of 40 μM MB delivered to each limb via 2 × 10 μL injections). After a post-injection interval ranging from 10 min to 18 h, muscles were dissected and analyzed as described above. Plots of MB fluorescence vs. time were used to estimate the time course of MB removal from the anterior compartment. These measurements yielded a time constant of ~4 h. By comparison, intravenous administration of MB in humans revealed multi-compartmental pharmacokinetics with a terminal plasma half-life of 5–7 h (Peter et al., 2000). Calculations using time constants ranging from 4 to 7 h suggest a steady-state total [MB] of 3–5 μM, consistent with the range of values measured above for pump-infused muscles. These values for total [MB] include MB in both extracellular and intracellular compartments. To estimate the intracellular/extracellular distribution of MB, living EDL muscles were incubated in a large volume of 5 μM MB for 3 h, then washed, fixed and analyzed as above. The total concentration of MB in muscle was at least 5× the bath concentration. This evidence that muscle cells accumulate MB is consistent with O'Leary et al.'s (2010) report that MB injected intraperitoneally accumulated in mouse CNS to concentrations N10 × greater than that in plasma. MB has been used to stain peripheral nerve fibers, including motor terminals (Waerhaug, 1992; reviewed in Oz et al., 2011), but under conditions of our experiments MB accumulation in muscle was much more evident than accumulation in nerve fibers in fixed tissue. Assuming that 14.4% of wet muscle weight is extracellular fluid (Sheff and Zacks, 1982), and that the intracellular concentration was 5× that in extracellular fluid, these results suggest that the 2–8 × 10−6 μmol/ mg total MB estimated above for pump-infused muscles corresponds to an intracellular concentration of 2.3–9.1 μM and an extracellular concentration of 0.45–1.8 μM. Each of these values includes both free and bound MB in these compartments. MB binds to albumin as well as to a variety of intracellular components, and accumulates within mitochondria (reviewed by Oz et al., 2011). References Atamna, H., Nguyen, A., Schultz, C., Boyle, K., Newberry, J., Kato, H., Ames, B.N., 2008. Methylene blue delays cellular senescence and enhances key mitochondrial biochemical pathways. FASEB J. 22, 703–712. Audet, J.N., Soucy, G., Julien, J.P., 2012. Methylene blue administration fails to confer neuroprotection in two amyotrophic lateral sclerosis mouse models. Neuroscience 209, 136–143. Augusto, V., Padovani, C.R., Campos, G.E.R., 2004. Skeletal muscle fiber types in C57BL6J mice. Braz. J. Morphol. Sci. 21, 89–94. Barber, S.C., Shaw, P.J., 2010. Oxidative stress in ALS: key role in motor neuron injury and therapeutic target. Free Radic. Biol. Med. 48, 629–641. Barrett, E.F., Barrett, J.N., David, G., 2011. Mitochondria in motor nerve terminals: function in health and in mutant superoxide dismutase 1 mouse models of familial ALS. J. Bioenerg. Biomembr. 43, 581–586. Beirowski, B., Berek, L., Adalbert, R., Wagner, D., Grumme, D.S., Addicks, K., Ribchester, R.R., Coleman, M.P., 2004. Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice. J. Neurosci. Methods 134, 23–35. Burke, R.E., 2004. The structure and function of motor units. In: Engeland, A.G., FranziniArmstrong, C. (Eds.), Myology, third ed. McGraw-Hill, New York, pp. 104–118. Carrasco, D.I., Bichler, E.K., Rich, M.M., Wang, X., Seburn, K.L., Pinter, M.J., 2012. Motor terminal degeneration unaffected by activity changes in SOD1G93A mice; a possible role for glycolysis. Neurobiol. Dis. 48, 132–140. Cozzolino, M., Carri, M.T., 2012. Mitochondrial dysfunction in ALS. Prog. Neurobiol. 97, 54–66. Dadon-Nachum, M., Melamed, E., Offen, D., 2011. The “dying-back” phenomenon of motor neurons in ALS. J. Mol. Neurosci. 43, 470–477. Daudt III, D.R., Mueller, B., Park, Y.H., Wen, Y., Yorio, T., 2012. Methylene blue protects primary rat retinal ganglion cells from cellular senescence. Invest. Ophthalmol. Vis. Sci. 53, 4657–4667.

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