Evaluation of Functional and Structural Alterations in ...

Original Paper Accepted after revision: January 10, 2011 Published online: April 14, 2011

Cells Tissues Organs DOI: 10.1159/000324148

Evaluation of Functional and Structural Alterations in Muscle Tissue after Short-Term Cold Storage in a New Tissue Preservation Solution Timo Wille a Sascha Gonder a Horst Thiermann a Thomas Seeger a Ursula Rauen b Franz Worek a  

 

 

 

 

 

a Bundeswehr Institute of Pharmacology and Toxicology, Munich, and b Institute of Physiological Chemistry, University Hospital, Essen, Germany  

 

Key Words Cold storage ⴢ Electrophysiology ⴢ Muscle preparations ⴢ Muscle force ⴢ Preservation solution ⴢ Mouse

Abstract Storage of muscle preparations in vitro is required for the diagnosis of neuromuscular disorders and for electrophysiological tests. The current standard protocols for muscle storage or transport, i.e. placement on 0.9% NaCl-moistened gauze, lead to impaired function and structural alterations. For other tissues, however, improved preservation methods and solutions have recently been described. In this study, functional and structural alterations in the murine diaphragm were compared after storage on 0.9% NaCl-moistened gauze and after storage in different modifications of the new vascular preservation solution TiProtec쏐. Muscle force generation after nerve stimulation, histological parameters and ATP levels were investigated after 2.5 h of cold storage at 4 ° C in the different media and 0.5 h of rewarming at 25 ° C in Tyrode buffer. Murine diaphragms were injured during cold storage and rewarming, with the degree of the al 

 

 

 

Dr. Ursula Rauen is a consultant of Dr. Franz Köhler Chemie, Bensheim, Germany. The study was funded by the German Ministry of Defence. TiProtec was provided by Dr. Franz Köhler Chemie for the preparation of the modified solution 1.

© 2011 S. Karger AG, Basel 1422–6405/11/0000–0000$38.00/0 Fax +41 61 306 12 34 E-Mail [email protected] www.karger.com

Accessible online at: www.karger.com/cto

teration being dependent on the type of solution used. There were no histological alterations and no caspase 3 activation in all groups. In contrast, diaphragms stored in the modified TiProtec solution showed markedly better performance concerning force generation after nerve stimulation (7.1 8 1.1 cN ⴢ s) as well as higher ATP content (2.4 8 0.7 ␮mol/g) and were superior to storage on 0.9% NaCl-moistened gauze (1.4 8 0.4 cN ⴢ s; 0.3 8 0.1 ␮mol/g). In conclusion, the modified TiProtec preservation solution showed promising results for short-term cold storage of murine diaphragms. For further evaluation, the transferability of these positive findings to storage conditions for muscles of other species, especially human muscle tissue, needs to be investigated. Copyright © 2011 S. Karger AG, Basel

Introduction

For the diagnosis of neuromuscular diseases and for electrophysiological tests, the current standard protocols for the transport and preparation of muscle specimens prescribe the deposition of the muscles on 0.9% NaCl-

Abbreviations used in this paper

UW HTK

University of Wisconsin histidine-tryptophan-ketoglutarate

Dr. Timo Wille Institut für Pharmakologie und Toxikologie der Bundeswehr Neuherbergstrasse 11 DE–80937 Munich (Germany) Tel. +49 89 3168 2305, E-Mail TimoWille @ Bundeswehr.org

moistened gauze in a Petri dish at low temperatures [Dubo-witz and Sewry, 2007], on damp gauze [Bergmann et al., 2009; Stanley et al., 2009] or even on dry gauze [Stanley et al., 2009]. In order to investigate the effects of new pharmacological approaches to restore impaired neuromuscular functions, e.g. for the development of novel reactivators of acetylcholinesterase inhibited by organophosphorus compounds, intercostal or diaphragm muscle specimens of animals and humans are required [Wolthuis et al., 1981; Thiermann et al., 2005; Seeger et al., 2007; Thiermann et al., 2010]. Especially human muscle tissue is regularly dissected in operation theaters, which are located far away from the respective diagnostic or research laboratory. Accordingly, there is an urgent need to optimize transport conditions as an impaired membrane potential [Oredsson et al., 1993] and decreased response to electrophysiological stimuli [van der Heijden et al., 2000] with subsequent loss of function and structural integrity of specimens were detected after shortterm cold storage [van der Heijden et al., 2000; Wagh et al., 2000]. So far, only few studies have been published in which muscle biopsies showed promising results regarding (ultra)structural alterations after 1–5 days when stored in a new tissue medium [Sandberg et al., 2007]. Finally, improved muscle preservation might optimize clinical applications in reconstructive and plastic surgery by the advancement of contractile and morphological properties and by the minimization of necrosis and inflammation [Wagh et al., 2000; de With et al., 2009]. Until recently, it has generally been thought that hypothermia inhibits the Na+/K+-ATPase with sodium influx and subsequent cell swelling. The current standard protocols for muscle preservation were therefore designed to counteract these alterations in cellular ion homeostasis. However, recent studies have shown that various cell types do not show an increase in intracellular sodium concentration [Gizewski et al., 1997; Frank et al., 2000]. Nevertheless, they suffer an injury which is mediated by irondependent formation of reactive oxygen species [Rauen et al., 1999, 2000; Huang and Salahudeen, 2002; Rauen and de Groot, 2004]; this injurious mechanism is not addressed by the current storage protocols although a similar type has been described for skeletal muscle tissue as well [de With et al., 2009]. A new cold storage solution for blood vessels, which takes this mechanism into consideration, has recently been introduced [Wille et al., 2008]. This N-acetylhistidine-buffered, potassium chloride-enriched and amino acid-fortified storage solution augmented with iron chelators (TiProtec쏐) showed substantial progress in long-term storage of porcine aortae 2

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regarding endothelial cell viability, mitochondrial membrane potential and inhibition of platelet adhesion to endothelial cells [Wille et al., 2008] as well as superior maintenance of vessel tone and vasoreactivity of rat [Zatschler et al., 2009] and human arteries [Garbe et al., 2011]. A slight modification of this vascular preservation solution containing higher concentrations of iron chelators has recently been successfully employed for the cold storage of human hepatocytes [Pless et al., 2011]. As protection of the mitochondrial membrane potential and muscle tone are also crucial parameters for muscle storage and as advantages of potassium-rich preservation solutions are still under debate, the aim of this study was to investigate the applicability of this newly developed solution, and the sodium-rich variant thereof, for storage and transport of skeletal muscle tissue, thereby possibly maintaining the structure and function of the specimens for further investigation or therapeutic interventions.

Materials and Methods Animals and Preparations Male NMRI mice (35 8 5 g; River, Sulzfeld, Germany) were anesthetized with carbon dioxide and killed by neck dislocation. Diaphragm hemispheres were rapidly excised. The experimental protocol has been approved based on the local animal protection act. Determination of Muscle Force Production Diaphragms were mounted vertically in a custom-made multiorgan bath with 12 glass chambers and fixed to force transducers (WPI, La Jolla, Calif., USA). The chambers were filled with Tyrode solution (25 ° C; table 1). After a 30-min adaptation period, the diaphragm hemispheres were stimulated with a pair of platinum electrodes, which had been placed parallel to the muscles. Electric field stimulation was performed with 50-␮s pulse widths and an amplitude of 2 A to achieve indirect stimulation. In order to confirm that muscle contraction by the electrical field stimulation technique was only induced via nerve trunks in the muscle preparations, the nicotinic antagonist pancuronium (10 ␮M) was added prior to completion of the test. Maintenance of force generation after direct stimulation in the absence of force generation after indirect stimulation proved indirect stimulation during the test period. Isometric muscle force production was determined and the amplified signals were recorded online (AcqKnowledge 3.8.1; Biopac Systems Inc., Goleta, Calif., USA). The areas under the curve of the respective force-time diagrams were used as test parameters. Tetanic trains with frequencies of 50 and 100 Hz for 1 s in 10-second intervals were applied for force generation throughout.  

 

Experimental Design Comparison of the Effects of the Preservation Solution on Muscle Function after Cold Storage. After fixation and accommodation of muscles in the organ baths, muscle force production after indirect stimulation (50 and 100 Hz) was recorded and used as control value. Immediately thereafter, the Tyrode solution was

Wille /Gonder /Thiermann /Seeger / Rauen /Worek  

 

 

 

 

 

Table 1. Composition of the solutions 1 and 2

Solutions

Cl– ␣-Ketoglutarate Aspartate HCO3– H2PO4– Na+ K+ Mg2+ Ca2+ N-Acetylhistidine Glycine Alanine Tryptophan Sucrose Glucose Deferoxamine LK 614 pH Osmolarity, mosm/l

1

2

103.1 2 5 – 1 16 93 8 0.05 30 10 5 2 20 10 0.5 0.017 7.0 306

103.1 2 5 – 1 103 6 8 0.05 30 10 5 2 20 10 0.5 0.017 7.0 306

0.9% NaCl

Tyrode solution

154 – – – – 154 – – – – – – – – – – – 5.9 309

149 – – 24 – 136 5.4 1 1.8 – – – – – 10 – – 7.4* 327

The concentrations of all compounds are given in mM. * Tyrode solution was aerated with 95% O2 and 5% CO2 (5 ml/ min).

solute values were compared to those determined in the comparison of solutions 1 and 2 before cold storage of the diaphragms as respective controls. ATP Assay Diaphragms were stored in solution 1 or 2, or in 0.9% NaClmoistened gauze, respectively, for 2.5 h at 4 ° C and were rewarmed for 0.5 h at 25 ° C in Tyrode solution. Subsequently, diaphragms were snap-frozen and kept at –80 ° C for further processing. For analysis of ATP, the frozen diaphragms were put into 5 ml of boiling Tris buffer (100 mM Tris and 4 mM EDTA, pH 7.75) in order to inactivate ATPase [Yang et al., 2002]. Then tissues were homogenized with an Ultra-Turrax (30 s, 13,500 rpm; IKA-Werke, Staufen, Germany) and diluted in another 5 ml of boiling Tris buffer. Further testing was conducted with an ATP bioluminescence assay kit (CLS II; Roche Diagnostics, Mannheim, Germany).  

 

 

 

 

 

Weight Changes in the Specimen and Histology Diaphragms of mice were excised, weighed on a precision balance (Sartorius AC210S, Göttingen, Germany), stored for 2.5 h in solution 1, solution 2 or in moist gauze (0.9% NaCl), respectively, at 4 ° C and weighed immediately after cold storage. Subsequently, diaphragms were rewarmed in Tyrode buffer for 0.5 h at 25 ° C and weighed again. Then, the diaphragms were fixed in 4% paraformaldehyde for at least 4 h. Thereafter, preparations were stained with hematoxylin and eosin for the assessment of morphological changes of nuclei and muscle fiber thickness. Cleaved caspase 3 was assessed by a monoclonal antibody assay (apoptosis marker: SignalStain Cleaved caspase 3; Cell Signaling Technology, Danvers, Mass., USA). A Zeiss Axio Imager.Z2 (Zeiss, Jena, Germany) and TissueFaxs soft- and hardware (Tissuegnostics, Vienna, Austria) were used for recording.  

 

 

 

removed and a slightly modified, pre-cooled (4 ° C) version of the new vascular preservation solution TiProtec (kindly provided by Dr. Franz Köhler Chemie, Bensheim, Germany; modification: 500 ␮ M instead of 82 ␮ M deferoxamine), being identical to the solution used for human hepatocyte storage [Pless et al., 2011] (solution 1) or a sodium-rich version thereof (solution 2; both prepared in the Institute of Physiological Chemistry, Essen, Germany; table 1), was filled into the chamber, respectively, and kept at 4 ° C for 2.5 h, thereby simulating cold storage and transport conditions of the diaphragms. Then the preservation solutions were removed, the diaphragms were washed and circumfused with cold aerated Tyrode solution (table 1), and subsequently rewarmed for 0.5 h at 25 ° C. Finally, force generation after indirect stimulation was recorded and compared to the control values which had been recorded prior to cold storage. Comparison of Different Cold Storage Methods. To simulate the situation of muscle storage and transport, diaphragms were mounted in Petri dishes immediately after removal from mice. One group was stored on 0.9% NaCl-moistened gauze (DeltaSelect, Munich, Germany), and the other group in solution 1 for 2.5 h at 4 ° C followed by 0.5 h of rewarming in Tyrode solution. Afterwards, muscles were mounted in the organ baths as described above. After a 30-min accommodation period, muscle force production after indirect stimulation was recorded. Absolute values from muscle force time diagrams were compared. Due to the experimental procedure, it was not possible to record control values prior to the use of the various solutions, and thus ab-

Statistics The values are given as means 8 SEM throughout. Student’s t test was used for statistical analyses of muscle force production experiments, with p ! 0.05 indicating a significant difference. To analyze effects of preservation solutions on ATP content and wet weight, intergroup differences were tested using a Kruskal-Wallis test with Dunn’s post test. A value of p ! 0.05 was taken to indicate a significant difference.

Muscle Cold Storage

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Results

Effect of the Preservation Solutions 1 and 2 on Force Generation in the Organ Bath Diaphragms in the organ baths were exposed to the TiProtec derivative solution 1 or the sodium-rich variant thereof, solution 2, for 2.5 h at 4 ° C, and were subsequently rewarmed in Tyrode buffer for 0.5 h at 25 ° C. Muscle force production of diaphragms stored in solution 1 showed a tendency to lower force production compared to the control values before storage after stimulation with 50 and 100 Hz (fig. 1). However, diaphragms stored in the sodium-rich solution 2 showed a significantly stronger decrease compared to the potassium-rich solution 1 at  

 

 

 

3

50 Hz

100 Hz

50 Hz

*

*

100 Hz

10

*

8

80

Force, AUC (cN · s)

Normalized force, AUC (%)

100

60 40 20

* 6 4 2

0 1

2

1

0

2

C

Solutions

S1

C

0.9% NaCl

S1

0.9% NaCl

Fig. 1. Muscle force generation of mouse diaphragms after 2.5 h

Fig. 2. Muscle force generation of unstored mouse diaphragms

cold storage (4 ° C) in solution 1 (n = 24) or solution 2 (n = 19) and 0.5 h rewarming in Tyrode buffer followed by tetanic stimulation with 50 and 100 Hz and a pulse width of 50 ␮s. Data were analyzed as time force diagram (area under curve, AUC) and related to the non-stored control values at the beginning of the test. Results are expressed as relative change 8 SEM of the muscle force compared to the control data. * p ! 0.005 vs. solution 2.

(control, C; n = 43) and after 2.5 h of cold storage (4 ° C) in solution 1 (S1; n = 22) or on moist gauze (0.9% NaCl; n = 23) and 0.5 h rewarming in Tyrode solution followed by tetanic stimulation with 50 and 100 Hz and a pulse width of 50 ␮s. Data were analyzed as time force diagram (area under curve, AUC). In these cases, muscle force was analyzed as absolute force generation, and results are expressed as absolute changes 8 SEM of the muscle force. * p ! 0.0001 vs. 0.9% NaCl-moistened gauze (0.9% NaCl).

 

 

 

Color version available online

 

a

b

Fig. 3. Tetanic trains of murine diaphragms immediately after preparation (a) and stimulation with 20, 50 or 100 Hz (50 ␮s pulse width) and after 2.5 h of storage of the respective diaphragms in solution 1 at 4 ° C and rewarming in Tyrode for 0.5 h at 25 ° C (b).  

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a Immediately after preparation, a short maximum contraction is detectable followed by a decrease in muscle force. b After cold storage in solution 1 and rewarming, the diaphragms showed a stable tetanus during the whole phase of stimulation.

Wille /Gonder /Thiermann /Seeger / Rauen /Worek  

 

 

 

 

 

both frequencies. The loss of contractility of diaphragms stored in solution 2 was too strong and solution 2 could therefore not be used as preservation solution. Thus, solution 1 was used in the subsequent experiments to simulate the transport conditions. Effect of Preservation Solution 1 and 0.9% NaCl-Moistened Gauze on Force Generation Explicit differences in force generation were found when diaphragms were placed in a Petri dish filled with solution 1 or stored on a 0.9% NaCl-moistened gauze for 2.5 h at 4 ° C, respectively, and subsequently rewarmed. Absolute force generation was significantly higher in diaphragms stored in solution 1 (approximately 80% of control values for unstored samples for both frequencies) compared to segments stored on moist gauze (fig. 2). Furthermore, the force of diaphragms stored in solution 1 was stable from 0.5 to 2.5 h (range: 15%) after rewarming at 25 ° C in Tyrode solution, whereas diaphragms stored on moist gauze showed a tendency to unstable force generation with an increase in force from 0.5 to 2 h and a subsequent decrease from 2 to 2.5 h (range 60%) compared to data immediately after cold storage.  

 

 

three groups after preparation as well as after rewarming (fig.  5). Immediately after cold storage on 0.9% NaClmoistened gauze, however, a decrease in the weight of each segment of about 20% was noted, whereas the wet weights of segments in solutions 1 and 2 did not change during cold storage. ATP The luciferase bioluminescence assay showed the highest ATP content in diaphragms that belonged to the unstored control group (fig.  6). Lower ATP levels were found in all diaphragms that had been subjected to cold storage in 0.9% NaCl-moistened gauze or solutions 1 and 2. After cold storage/rewarming, ATP levels of diaphragms immersed in solutions 1 and 2 were significantly higher compared to those obtained with 0.9% NaClmoistened gauze.

 

Qualitative Characteristics As a further aspect, force curves showed an unstable tetanus immediately after preparation suggesting fatigue (15 of 37 preparations with subsequent storage in solution 1; fig. 3, original tracing) in some preparations. If the same hemispheres of the diaphragm were stored in solution 1 for 2.5 h at 4 ° C, muscles (13 of 15) recovered and showed a stable tetanus for the rest of the experiment. This phenomenon was unique for diaphragms stored in solution 1 and not detectable after storage in solution 2 or in moist gauze.  

 

Histology Staining the specimens with hematoxylin and eosin did not reveal any obvious differences between unstored control, solution 1, solution 2 or 0.9% NaCl-moistened gauze (fig. 4). No morphological alterations in nuclei were found and the fiber size was similar in all conditions tested. No caspase 3 was detectable.

Discussion

The present results show that murine diaphragms suffer an injury displayed as functional impairment during cold storage and subsequent rewarming, i.e. typical conditions of a tissue transport. A new mechanism-based tissue preservation solution provides protection against this injury so that maintenance of force generation in murine diaphragms can be markedly improved. Cold Storage Injury Cold storage injury is a compound injury elicited by (i) hypoxia; (ii) hypothermia (used for cellular protection), and (iii) potentially by the preservation solution used [Rauen and de Groot, 2004]. The extent to which each injurious factor is involved depends on the cell type, tissue, oxygen availability, size of tissue as well as storage conditions.

Weight Changes If diaphragms were placed in sealed Petri dishes in solutions 1 or 2 or stored on a 0.9% NaCl-moistened gauze for 2.5 h at 4 ° C, respectively, and were subsequently rewarmed in Tyrode buffer for 0.5 h at 25 ° C, wet weights of the single segments did not vary significantly among the

Main Injurious Mechanisms and Counterbalancing Ingredients of the Solution Although hypothermia is employed to reduce metabolism, inhibit ATP decline and delay injurious processes, hypothermia also elicits cell injury [Hochachka, 1986; Blankensteijn and Terpstra, 1991; Rauen et al., 1999, 2003; Salahudeen et al., 2003; Rauen and de Groot, 2004]. Earlier hypotheses suggested inhibition of Na+/K+-ATPase with subsequent intracellular accumulation of sodium and chloride and cell swelling as major inducers of coldinduced cell injury [Hochachka, 1986; Blankensteijn and Terpstra, 1991], and according to these alterations of ion

Muscle Cold Storage

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Color version available online

50 μm

a

Fig. 4. Histological assessment of control diaphragm (a) and diaphragms after cold storage for 2.5 h in solution 1 (b), solution 2 (c) or on 0.9% NaCl-moistened gauze (d) at 4 ° C with subsequent rewarming for 0.5 h at 25 ° C in Tyrode solution. After rewarming, the diaphragms were fixed and stained with hematoxylin and the immunhistochemical marker for active caspase 3. No nuclear changes or immunhistochemical alterations were detectable. The pictures shown are representative for the group.  

 

 

0.15 Wet weight (g)

Wet weight (g)

0.15 0.10

0.05

0 In

a

d

c

a iti

l

r te Af r a g e o st

r te n g Af r m i a w re

0.15 Wet weight (g)

 

b

0.10 0.05 0 In

b

a iti

l

r te Af r a g e o st

r te n g Af r m i a w re

0.10 0.05 0 In

iti

al

c

r te Af r a g e o st

r te n g Af r m i a w re

Fig. 5. Wet weight of murine diaphragms immediately after excision (initial), after 2.5 h of storage (after stor-

age) and after 0.5 h rewarming in Tyrode buffer (after rewarming). Data are expressed as wet weight change of each segment for storage in solution 1 (a), solution 2 (b) or on 0.9% NaCl-moistened gauze (c).

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ATP content (µmol/g diaphragm)

4

3

* *

2

1

0

C

0.9% NaCl

S1

S2

Fig. 6. ATP content (␮mol/g diaphragm 8 SEM) in murine diaphragms of unstored control specimens (C) and after 2.5 h of cold storage (4 ° C ) in solution 1 (S1), solution 2 (S2) or on 0.9% NaClmoistened gauze (0.9% NaCl) and 0.5 h rewarming in Tyrode buffer. * p ! 0.05 vs. 0.9% NaCl-moistened gauze.  

 

homeostasis, the currently used preservation solutions, the University of Wisconsin (UW) solution [Belzer and Southard, 1988] and the histidine-tryptophan-ketoglutarate (HTK) solution [Bretschneider et al., 1988], were developed. Recent studies have challenged this view [Gizewski et al., 1997; Fuckert et al., 2000] and shown that an iron-dependent formation of reactive oxygen species constitutes the major factor in cold-induced injury of various cell types [Rauen et al., 1999, 2000; Salahudeen et al., 2003]. The formation of highly reactive species appears to be triggered by a cold-induced increase in the cellular chelatable (or ‘free’), ‘redox-active’ iron pool [Rauen et al., 1999, 2000; Huang and Salahudeen, 2002; Rauen and de Groot, 2004], and mitochondria have been shown to be the major target of the reactive species (see ‘ATP’ below) [Kerkweg et al., 2003; Rauen et al., 2003; Salahudeen et al., 2003]. A similar mechanism also appears to be at play during cold storage of muscle tissue [Magni et al., 1994; van der Heijden et al., 2000]. To stop this iron-dependent injury, the strong, hexadentate but poorly membranepermeable iron chelator deferoxamine and the smaller, aromatic and thus more lipophilic membrane-permeable iron chelator LK 614 are present in TiProtec and its variants used in this study. Due to the encouraging results with higher chelator concentrations in cold storage of isolated cells [Pless et al., 2011] and in muscle cold storage [Magni et al., 1994; van der Heijden et al., 2000], 500 ␮M deferoxamine were used in solutions 1 and 2 instead of 82 ␮M in TiProtec to ensure safe chelation. Current preservation methods (0.9% NaCl-moistened gauze) and soluMuscle Cold Storage

tions (HTK and UW) do not consider this mechanism as they originally do not contain iron chelators. TiProtec and the variants used in this study contain N-acetylhistidine as buffer because histidine, the main buffer system of the HTK preservation solution, and phosphate, the main buffer system of the UW solution, are excellent buffers but also exert some toxicity [Rauen et al., 2007]. Nacetylhistidine does not share this toxicity [Rauen et al., 2007]. TiProtec and its variants contain glycine and alanine to avoid the formation of non-specific pores resulting in sodium influx into the cells, a major mechanism of hypoxic injury [Carini et al., 1997; Frank et al., 2000; Rauen and de Groot, 2004]. Furthermore, TiProtec, its variants and the parent organ preservation solution Custodiol-N [Wu et al., 2009, 2011], contain tryptophan (considered to be a membrane-stabilizing compound [Bretschneider et al., 1988]) and ␣-ketoglutarate. Aspartate was added to allow, together with the latter, the provision of intermediates of the citric acid cycle [Wu et al., 2009]. The pH of the solutions is 7.0, as a mild acidosis shows protection against several types of cell injury [Koop and Piper, 1992; Nishimura et al., 1998]. The vascular preservation solution TiProtec contains glu-cose as a substrate for highly glycolytic endothelial cells [Mertens et al., 1990; Davidson and Duchen, 2007], and this was also employed in this study as a substrate for muscle cells [Hultman and Sjöholm, 1983], while current organ preservation solutions (UW and HTK) do not contain glucose. Using a very thin specimen as in the current study, lack of oxygen does probably not constitute a major problem so that the formation of cold-induced reactive oxygen species is likely to be the major injurious mechanism. The better protection by TiProtec variants may therefore most likely be ascribed to the iron chelators and the higher potassium concentration (fig. 1, 2). Sodium versus Potassium The finding that the potassium-rich solution 1 maintains the response to electrophysiological stimuli significantly better than the sodium-containing solution 2 (fig. 1) seems curious as it is described that the outflow of the glycolytic enzyme aldolase was higher in a potassiumrich storage solution in a rat diaphragm model [Zierler, 1956]. However, in that study, complex solutions with different ionic compositions were compared and did not clearly discriminate the effect of potassium. A benefit of a high potassium concentration was also observed during cold storage of porcine aortic segments [Wille et al., 2008] and rat mesenteric arteries [Zatschler et al., 2009]. The currently used HTK solution is potassium poor and Cells Tissues Organs

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might not be the first choice for muscle preservation, although it showed better results than potassium-rich UW solution in a rat skeletal muscle model [van der Heijden et al., 1999]. Possibly, a high extracellular potassium concentration during cold storage inhibits potassium efflux [Collins et al., 1969; D’Alessandro et al., 1994], which has been shown to induce apoptosis [Lang et al., 2005]. Solution 1 versus 0.9% NaCl-Moistened Gauze Storage on moist gauze has been historically used by pathologists to minimize liquid uptake into the tissue and to avoid ice crystallization when using a cryostat method for further processing of specimens [Dubowitz and Sewry, 2007; Bergmann et al., 2009]. However, a decrease in wet weight of 20% in diaphragms stored on 0.9% NaCl-moistened gauze was observed here (fig. 5), which is probably also not beneficial. Furthermore, in solutions 1 and 2 the anticipated water uptake during immersion storage was not observed. This is particularly remarkable in the sodium- and chloride-rich solution 2, where this finding contradicts the old hypothesis that cold storage leads to cell swelling [Hochachka, 1986; Blankensteijn and Terpstra, 1991] and might raise the question of whether usage of the preservation solutions UW and HTK to counteract water uptake is rational. As both force generation and reproducibility of electrophysiological parameters were far better after cold storage in solution 1 than after cold storage on moist gauze (fig. 2), the data suggest that storage in solution 1 is the preferable method. ATP It has been shown that mitochondria are the main target for cold-induced, iron-dependent injury [Kerkweg et al., 2003; Rauen et al., 2003; Rauen and de Groot, 2004; Rauen et al., 2006]. Here, the ATP content of muscles was clearly dependent on the method of cold storage, while solutions 1 and 2 did not differ significantly but showed a tendency to higher ATP values in the segments stored in solution 1 (fig.  6). While the latter is similar to the maintenance of the mitochondrial membrane potential in stored vessels [Wille et al., 2008], the former suggests that mitochondrial injury was predominantly inhibited by a compound present in both solutions, most probably by the iron chelators. Histology Histological parameters did not reveal any changes regarding nuclear morphology, fiber diameter and cleavage of caspase 3 (fig. 4) after storage in all solutions. This is possible as the development of morphological alterations 8

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might need a longer cold storage time than functional impairment [Oredsson et al., 1993; van der Heijden et al., 2000], which is most probably beyond the test period examined here. Limits of the Study In this assay, the effectiveness of the different storage solutions was scrutinized using very thin muscle samples and showed a clear superiority of solution 1. For larger muscles, where higher buffering power is required, the parent organ preservation solution Custodiol-N, which is based on the same principles as the solution used here, might be an alternative.

Conclusion

The modified TiProtec solution 1 improves the preservation of murine diaphragms for electrophysiological tests and might be a promising approach for further applications in the diagnostic field as well as in the therapeutic field of reconstructive and transplantation medicine. For further evaluation, the transferability of these positive findings to muscles of other species, especially human muscle tissue, needs to be investigated.

Acknowledgments The authors are grateful to S. Baldauf and S. Müller for their expert technical assistance.

References

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Wille /Gonder /Thiermann /Seeger / Rauen /Worek  

 

 

 

 

 

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