Neuromuscular adaptations to cross-reinnervation in ‘1% and 2%mo-old Fischer 344 rats KATHRYN
I. CLARK
AND
TIMOTHY
Department of Kinesiology and Institute Ann Arbor, Michigan 48109-2214
P. WHITE of Gerontology, The University of Michigan,
CLARK, KATHRYN I., AND TIMOTHY P. WHITE. Neuromuscular adaptations to cross-reinnervation in 12- and 29-mo-old Fischer 344 rats. Am. J. Physiol. 260 (Cell Physiol. 29): C96-
C103, 1991.-The aim was to test hypotheses regarding the adaptive responseof the extensor digitorum longus (EDL) muscleof l2- and 29-mo rats following denervation and crossreinnervation by the soleusnerve. The massof cross-reinnervated EDL musclewas87 and 86% of self-reinnervated control values in 12-mo (99 t 3 mg) and 29-mo (74 t 3 mg) rats, respectively. Cross-reinnervated EDL fiber area was 56 and 67% of self-reinnervated values in 12-mo (1,733 k 253 pm2) and 29-mo (1,264 t 71 pm2) rats, respectively. Cross-reinnervation increasedthe density of neural contact 26% in 12-mo rats and decreaseddensity by 50% in 29-mo animals.In 12-mo rats 17% of motor end plates (MEP) were void of terminal nervesfollowing cross-reinnervationcomparedwith 48% in 29mo rats. In cross-reinnervated muscles,slow myosin heavy chain (MHC) was65 t 9 and 25 t 3% of total MHC in 12- and 29-morats, respectively. The percentageof type I fibers derived histochemicallywas65 t 8% in 12-morats and 18 t 1% in 29mo rats. In conclusion,there is an age-associateddecreasein the ability of neuronsto reinnervate the MEP area after nerve section. The conversion of fiber type in innervated fibers in responseto cross-reinnervationmay not differ due to age. aging; skeletal muscle; myosin heavy chain; histochemistry; fiber type; motor end plate; terminal nerves; reinnervation; denervation
of terminal innervation in skeletal muscle changes throughout life due to an ongoing process of degeneration of terminal axons followed by sprouting and reinnervation by remaining terminal nerves (2). Ageassociated decrements in neuronal proteosynthesis (19) and axoplasmic transport (14) may result in a decline in the ability of terminal neurons to reinnervate following degeneration. With aging there is a diminished or slowed response of axons to regenerate or reinnervate the motor end-plate (MEP) region following perturbation ( 11, 27). Aging of the peripheral nervous system is also associated with decreased efficacy of transmission as a result of segmental demyelination (6) that results in decreased action potential conduction velocity (14). The stimulation rate at which action potential propagation failure occurs is also lower in old animals than in adults (30), which results in the inability of all fibers in the motor unit to receive an adequate stimulus (14). The consequence of impaired reinnervation would be a reduced nerve-muscle interaction and eventual denervation of THE CONFIGURATION
C96
individual muscle fibers that would be expressed as a decreased capacity to generate and maintain power. The purpose of this experiment was to compare in adult (12mo) and aged (29-mo) rats the ability of neurons to reinnervate the MEP following nerve section and the ability of the muscle fibers to adapt to the reinnervating neuron. Cross-reinnervation of skeletal muscle was used in the present study because it allowed for the assessment of both morphological parameters of reinnervation of the MEP and adaptation of innervated muscle fibers to atypical innervation. Cross-reinnervation experiments have typically innervated a predominantly slow muscle with a nerve that normally innervates a fast muscle and vice versa (4). After cross-reinnervation, adaptations occur in molecular, biochemical, and morphological characteristics of muscle fibers leading to altered function that resembles the muscle originally innervated by the nerve (1, 8, 15, 21, 34). The specific aim of the experiment was to study in adult and aged rats MEPs and muscle fibers in unoperated control muscles, muscles in which the nerve was severed and reimplanted (self-reinnervated), and muscles in which the extensor digitorum longus (EDL) nerve was severed and the soleus nerve was implanted (cross-reinnervated). Because of the aforementioned impairments in the peripheral nervous system with age, we hypothesized that the process of reinnervation and the adaptation to reinnervation following cross-reinnervation would be impaired in muscles of aged animals compared to those of adults. Experiments were designed to test the following hypotheses: compared with that in 12-mo rats, cross-reinnervation of the EDL muscle with the soleus nerve in 29-mo rats will result in 1) a smaller fiber crosssectional area, 2) fewer terminal axons per motor end plate, 3) a smaller percentage of type I fibers determined histochemically, and 4) a smaller percentage of the slow isoform of myosin heavy chain. METHODS
Specific pathogen-free male Fischer 344 rats were obtained from the Nationa 1 Institute on Aging sponsored colony at Harlan Industries (Indianapolis, IN) at 10 (n = 18) and 27 (n = 36) months of age. Rats were maintained on laboratory chow (Purina Lab Chow 5001) and water ad libitum, housed two per cage on bedding of ground corn cobs (Bed-O-Cobs), and kept on a l2:12 h
0363-6143/91 $1.50 Copyright 0 1991 the American Physiological Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 13, 2019.
CROSS-REINNERVATION
OF
SKELETAL
light-dark cycle in a specific pathogen-free barrier facility at the University of Michigan. All procedures were in accordance with the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were approved by the University Committee on Use and Care of Animals. Rats were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg), and cross-reinnervation operations were performed unilaterally by implantation of the soleus nerve into the EDL muscle. The tibia1 nerve was blunt dissected free of its connective tissue distal to the point where the muscular branches originate (16). The EDL muscle was temporarily denervated as the deep peroneal nerve was cut free and folded back between the peroneus digiti quarti and the peroneus brevis muscles. By use of an adaptation of a method from White et al. (32), the soleus nerve was severed at the point it entered the soleus muscle. The soleus nerve was dissected free, passed latera1 to the peroneus muscles, and implanted into the EDL muscle. The nerve implant was held with a 7-O silk purse-string suture at the location previously of the most proximal branch of the EDL nerve. Contralateral legs provided self-reinnervated tissues in which the nerves to the soleus and EDL muscles were severed and reimplanted orthotopically. Drinking water was supplemented with tetracycline 1 day before the operation and for 2 days afterward to prevent infection. An additional cohort of twelve 27-mo animals and six IO-mo animals provided control muscles that received no surgical intervention. With the expected 50% mortality rate of 27-mo rats (33), the design yielded 18 muscles in the following group designations at two ages: 1) EDL muscles crossreinnervated with the soleus nerve (cross-reinnervated), 2) EDL muscles in which the EDL nerve was severed and reimplanted (self-reinnervated), and 3 ) soleus muscles with a severed and reimplanted soleus nerve. The design also provided for 12 unoperated control soleus and EDL muscles at each age. Fifty-six days after operations, careful blunt dissection of the cross-reinnervated EDL muscle was performed and no evidence of self-reinnervation by the EDL nerve was found. After blunt dissection and cutting of the distal tendon of the EDL muscle, the viability of the soleus nerve innervating the EDL muscle was evaluated. All muscles were then excised, weighed, and prepared either for standard histochemistry and gel electrophoresis or for quantification of motor end plates and terminal axons. For histochemical preparation, a sample was cut from the full cross section of the muscle belly and frozen in isopentane cooled to -70°C by dry ice. The remaining piece of muscle was frozen for gel electrophoresis. Transverse sections (10 pm thick) were cut from the histochemical sample in a Damon IEC cryostat (Ames, Elkhart, IN). Serial sections were incubated for myofibrillar adenosinetriphosphatase (ATPase) activity at pH 10.3 and 4.6 (3) and for succinate dehydrogenase (SDH) activity (25). Approximately 500 fibers were studied in each muscle section. The activity of myosin ATPase at different pHs was used to differentiate fiber types (3), and cell size was quantified with planimetry (Bioquant image analvsis) in histochemical sections which had been in-
MUSCLE
IN
AGED
RATS
c97
cubated in alkaline pH. Type I fibers appeared light at pH 10.3 and dark at 4.6 and were dark following SDH incubation (3). Fibers that were dark at alkaline pH were classified as type II. These fibers were further classified as IIa if they appeared light at pH 4.6 and dark in SDH, as IIb if they were dark at pH 4.6 and light following SDH incubation, or as IIc if they remained dark at pH 4.6 and 4.3 (3). Direct determination of the relative proportions of slow and fast myosin heavy chain (MHC) was performed using sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) (13). Muscle samples were homogenized (1:lO wt/vol) in 62.5 mM tris(hydroxymethyl)aminomethane buffer (Tris, pH 6.8). After removal of an aliquot for determination of protein concentration (23), the remaining homogenate was combined with an equal volume of buffer, yielding a 1:20 tissue homogenate for electrophoresis. Homogenate (10 ~1) was electrophoresed on 5% SDS-PAGE for 12 h. Gels were scanned on an LKB Ultroscan XL laser densitometer (model 2202), and the slow and fast MHC peaks were quantified using an IBM AT microcomputer and Gelscan software (version 1.2). MEPs and terminal nerves were stained in 40-pmthick longitudinal sections using the method of Pestronk and Drachman (26). Sections were fixed in 1.4 M sodium sulfate (Na2S04) and incubated for 40 min at 37°C in a solution containing 10 mM Tris HCl, 1.1 mM Tris base, 6.7 mM calcium chloride (CaCl& 3.3 mM potassium ferrocyanide [ K4Fe( CN)6], 0.5 mM potassium ferricyanide [K3Fe(CN)s], 1% ethanol, and 0.5 mM 5-bromoindoxyl acetate to elucidate motor end plates, which appear light blue in this reaction. Sections were then dehydrated in 70 and 100% ethanol before being fixed to the slide with 0.5% (wt/vol) celloidin. The secured sections were fixed at room temperature in buffered formal-saline (pH 7.0) containing 3.7% formaldehyde, 14.5 mM sodium chloride (NaCl), 1.3 mM acid sodium monophosphate (NaH,PO& and 1.8 mM anhydrous disodium phosphate (Na,HPO,) and fixed again at 37°C in 0.6 M chloral hydrate before a 40-min incubation at 37°C in a solution containing 1.2 M silver nitrate (AgNO,) and 4 mM cupric sulfate (CuSOJ. Sections were developed in a solution containing 90 mM hydroquinone and 0.4 mM sodium sulfite ( NaS03), fixed in 0.2 M sodium thiosulfate (Na2S203), toned with 5 mM sodium tetrachloroaurate (NaAuC&), and fixed again with 0.2 M Na2S203. MEP and terminal nerve data were quantified in MEPs (n = 1,320) as follows: 1) MEP area, 2) the number of terminal nerves per MEP, 3) the summated lengths of all terminal nerves within a MEP, and 4) the ratio of the total nerve length to the MEP area as an indication of the density of neuronal contact (28). An additional sample of approximately 200 MEPs per muscle was used to quantify the percentage of MEPs that were void of terminal nerves. For the histochemical and MEP data the means derived from individual muscles were averaged, resulting in the mean t SE of the mean. A two-way analysis of variance was used to analyze the data for main effects of age and cross-reinnervation and for interactions between age and experimental treatment. The Bonferroni post
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 13, 2019.
C98
CROSS-REINNERVATION
OF
SKELETAL
hoc test for critical differences was used to compare individual means, and P 5 0.05 was considered significant.
IN
AGED
RATS
muscles of 12-mo rats was normally distributed over a wide range of fiber areas compared with other groups, with a small percentage of fibers < 1,000 pm2 (Fig. 1). In control EDL muscle there was an increase from 12 to 29 mo in the ratio of total terminal nerve length to MEP area (Fig. 2, A and C, Table 2), which indicates a higher density of neuronal contact at individual neuromuscular junctions in muscles from 29-mo rats. The higher density from 12 to 29 mo of age resulted from a 50% decrease in MEP area, a 23% decrease in terminal nerve number, and an 18% decline in total terminal nerve length (Table 2). Compared with control EDL muscles from 12-mo rats, cross-reinnervation resulted in a 26% increase in the density of neuronal contact caused by a 27% decrease in MEP area and no change in nerve terminal number nor total terminal nerve length (Fig. 2B, Table 2). There were no differences in any of the MEP and terminal nerve parameters between self- and cross-reinnervated muscles in 12-mo rats. Compared with control EDL muscles in 29-mo animals, cross-reinnervation resulted in a 50% decrease in density due to a 48% decrease in terminal nerve number and 44% decrease in total terminal nerve length with no change in MEP area (Fig. 20, Table 2). In 29-mo rats, differences in MEP area between self- and cross-reinnervated muscles accounted for the increased density of neuronal contact in crosscompared with self-reinnervated muscles. The number of noninnervated MEPs increased with age in control EDL muscles (1 vs. 6% in 12 and 29 mo, respectively). After self- and cross-reinnervation, the number of noninnervated MEPs increased compared with control EDL muscle in both l2- and 29-mo rats. The magnitude of the increase was greater in 29-mo rats (17% in 12 mo 1 anu average Jzoer cross-sectzonal area
RESULTS
By 29 mo of age, body mass decreased 23% from the 12mo value of 438 t 7 g (Table 1). There was no difference in body weight due to experimental intervention at either age. Fifty-six days after operations, self-reinnervated EDL muscle mass was 41 and 40% less than the control values of 162 t 3 and 112 t 3 mg for 12- and 29-mo animals, respectively (Table 1). Mass of the cross-reinnervated EDL muscle was 13 and 14% lower than the self-reinnervated value of 98 t 3 and 73 t 3 mg from 12- and 29mo animals, respectively (Table 1). Protein concentration of muscle did not differ due to age but decreased 12 and 16% compared with control EDL muscle in self- and cross-reinnervated muscles, respectively (Table 1). There was no difference in protein concentration between the self- and cross-reinnervated groups. Fiber cross-sectional area (CSA) of self-reinnervated EDL muscles decreased 30% from the control value of 2,490 t 310 pm2 in 12-mo rats and decreased 32% from the control value of 1,866 t 195 pm2 in 29-mo rats (Table 1). The average fiber CSA of cross-reinnervated muscles decreased 44 and 33% compared with the fiber area of self-reinnervated muscles in l2- and 29-mo rats, respectively (Fig. 1, Table 1). However, most of fibers from self- and cross-reinnervated EDL muscles of 29-mo rats were
I. CLARK
AND
TIMOTHY
Department of Kinesiology and Institute Ann Arbor, Michigan 48109-2214
P. WHITE of Gerontology, The University of Michigan,
CLARK, KATHRYN I., AND TIMOTHY P. WHITE. Neuromuscular adaptations to cross-reinnervation in 12- and 29-mo-old Fischer 344 rats. Am. J. Physiol. 260 (Cell Physiol. 29): C96-
C103, 1991.-The aim was to test hypotheses regarding the adaptive responseof the extensor digitorum longus (EDL) muscleof l2- and 29-mo rats following denervation and crossreinnervation by the soleusnerve. The massof cross-reinnervated EDL musclewas87 and 86% of self-reinnervated control values in 12-mo (99 t 3 mg) and 29-mo (74 t 3 mg) rats, respectively. Cross-reinnervated EDL fiber area was 56 and 67% of self-reinnervated values in 12-mo (1,733 k 253 pm2) and 29-mo (1,264 t 71 pm2) rats, respectively. Cross-reinnervation increasedthe density of neural contact 26% in 12-mo rats and decreaseddensity by 50% in 29-mo animals.In 12-mo rats 17% of motor end plates (MEP) were void of terminal nervesfollowing cross-reinnervationcomparedwith 48% in 29mo rats. In cross-reinnervated muscles,slow myosin heavy chain (MHC) was65 t 9 and 25 t 3% of total MHC in 12- and 29-morats, respectively. The percentageof type I fibers derived histochemicallywas65 t 8% in 12-morats and 18 t 1% in 29mo rats. In conclusion,there is an age-associateddecreasein the ability of neuronsto reinnervate the MEP area after nerve section. The conversion of fiber type in innervated fibers in responseto cross-reinnervationmay not differ due to age. aging; skeletal muscle; myosin heavy chain; histochemistry; fiber type; motor end plate; terminal nerves; reinnervation; denervation
of terminal innervation in skeletal muscle changes throughout life due to an ongoing process of degeneration of terminal axons followed by sprouting and reinnervation by remaining terminal nerves (2). Ageassociated decrements in neuronal proteosynthesis (19) and axoplasmic transport (14) may result in a decline in the ability of terminal neurons to reinnervate following degeneration. With aging there is a diminished or slowed response of axons to regenerate or reinnervate the motor end-plate (MEP) region following perturbation ( 11, 27). Aging of the peripheral nervous system is also associated with decreased efficacy of transmission as a result of segmental demyelination (6) that results in decreased action potential conduction velocity (14). The stimulation rate at which action potential propagation failure occurs is also lower in old animals than in adults (30), which results in the inability of all fibers in the motor unit to receive an adequate stimulus (14). The consequence of impaired reinnervation would be a reduced nerve-muscle interaction and eventual denervation of THE CONFIGURATION
C96
individual muscle fibers that would be expressed as a decreased capacity to generate and maintain power. The purpose of this experiment was to compare in adult (12mo) and aged (29-mo) rats the ability of neurons to reinnervate the MEP following nerve section and the ability of the muscle fibers to adapt to the reinnervating neuron. Cross-reinnervation of skeletal muscle was used in the present study because it allowed for the assessment of both morphological parameters of reinnervation of the MEP and adaptation of innervated muscle fibers to atypical innervation. Cross-reinnervation experiments have typically innervated a predominantly slow muscle with a nerve that normally innervates a fast muscle and vice versa (4). After cross-reinnervation, adaptations occur in molecular, biochemical, and morphological characteristics of muscle fibers leading to altered function that resembles the muscle originally innervated by the nerve (1, 8, 15, 21, 34). The specific aim of the experiment was to study in adult and aged rats MEPs and muscle fibers in unoperated control muscles, muscles in which the nerve was severed and reimplanted (self-reinnervated), and muscles in which the extensor digitorum longus (EDL) nerve was severed and the soleus nerve was implanted (cross-reinnervated). Because of the aforementioned impairments in the peripheral nervous system with age, we hypothesized that the process of reinnervation and the adaptation to reinnervation following cross-reinnervation would be impaired in muscles of aged animals compared to those of adults. Experiments were designed to test the following hypotheses: compared with that in 12-mo rats, cross-reinnervation of the EDL muscle with the soleus nerve in 29-mo rats will result in 1) a smaller fiber crosssectional area, 2) fewer terminal axons per motor end plate, 3) a smaller percentage of type I fibers determined histochemically, and 4) a smaller percentage of the slow isoform of myosin heavy chain. METHODS
Specific pathogen-free male Fischer 344 rats were obtained from the Nationa 1 Institute on Aging sponsored colony at Harlan Industries (Indianapolis, IN) at 10 (n = 18) and 27 (n = 36) months of age. Rats were maintained on laboratory chow (Purina Lab Chow 5001) and water ad libitum, housed two per cage on bedding of ground corn cobs (Bed-O-Cobs), and kept on a l2:12 h
0363-6143/91 $1.50 Copyright 0 1991 the American Physiological Society
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 13, 2019.
CROSS-REINNERVATION
OF
SKELETAL
light-dark cycle in a specific pathogen-free barrier facility at the University of Michigan. All procedures were in accordance with the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were approved by the University Committee on Use and Care of Animals. Rats were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg), and cross-reinnervation operations were performed unilaterally by implantation of the soleus nerve into the EDL muscle. The tibia1 nerve was blunt dissected free of its connective tissue distal to the point where the muscular branches originate (16). The EDL muscle was temporarily denervated as the deep peroneal nerve was cut free and folded back between the peroneus digiti quarti and the peroneus brevis muscles. By use of an adaptation of a method from White et al. (32), the soleus nerve was severed at the point it entered the soleus muscle. The soleus nerve was dissected free, passed latera1 to the peroneus muscles, and implanted into the EDL muscle. The nerve implant was held with a 7-O silk purse-string suture at the location previously of the most proximal branch of the EDL nerve. Contralateral legs provided self-reinnervated tissues in which the nerves to the soleus and EDL muscles were severed and reimplanted orthotopically. Drinking water was supplemented with tetracycline 1 day before the operation and for 2 days afterward to prevent infection. An additional cohort of twelve 27-mo animals and six IO-mo animals provided control muscles that received no surgical intervention. With the expected 50% mortality rate of 27-mo rats (33), the design yielded 18 muscles in the following group designations at two ages: 1) EDL muscles crossreinnervated with the soleus nerve (cross-reinnervated), 2) EDL muscles in which the EDL nerve was severed and reimplanted (self-reinnervated), and 3 ) soleus muscles with a severed and reimplanted soleus nerve. The design also provided for 12 unoperated control soleus and EDL muscles at each age. Fifty-six days after operations, careful blunt dissection of the cross-reinnervated EDL muscle was performed and no evidence of self-reinnervation by the EDL nerve was found. After blunt dissection and cutting of the distal tendon of the EDL muscle, the viability of the soleus nerve innervating the EDL muscle was evaluated. All muscles were then excised, weighed, and prepared either for standard histochemistry and gel electrophoresis or for quantification of motor end plates and terminal axons. For histochemical preparation, a sample was cut from the full cross section of the muscle belly and frozen in isopentane cooled to -70°C by dry ice. The remaining piece of muscle was frozen for gel electrophoresis. Transverse sections (10 pm thick) were cut from the histochemical sample in a Damon IEC cryostat (Ames, Elkhart, IN). Serial sections were incubated for myofibrillar adenosinetriphosphatase (ATPase) activity at pH 10.3 and 4.6 (3) and for succinate dehydrogenase (SDH) activity (25). Approximately 500 fibers were studied in each muscle section. The activity of myosin ATPase at different pHs was used to differentiate fiber types (3), and cell size was quantified with planimetry (Bioquant image analvsis) in histochemical sections which had been in-
MUSCLE
IN
AGED
RATS
c97
cubated in alkaline pH. Type I fibers appeared light at pH 10.3 and dark at 4.6 and were dark following SDH incubation (3). Fibers that were dark at alkaline pH were classified as type II. These fibers were further classified as IIa if they appeared light at pH 4.6 and dark in SDH, as IIb if they were dark at pH 4.6 and light following SDH incubation, or as IIc if they remained dark at pH 4.6 and 4.3 (3). Direct determination of the relative proportions of slow and fast myosin heavy chain (MHC) was performed using sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE) (13). Muscle samples were homogenized (1:lO wt/vol) in 62.5 mM tris(hydroxymethyl)aminomethane buffer (Tris, pH 6.8). After removal of an aliquot for determination of protein concentration (23), the remaining homogenate was combined with an equal volume of buffer, yielding a 1:20 tissue homogenate for electrophoresis. Homogenate (10 ~1) was electrophoresed on 5% SDS-PAGE for 12 h. Gels were scanned on an LKB Ultroscan XL laser densitometer (model 2202), and the slow and fast MHC peaks were quantified using an IBM AT microcomputer and Gelscan software (version 1.2). MEPs and terminal nerves were stained in 40-pmthick longitudinal sections using the method of Pestronk and Drachman (26). Sections were fixed in 1.4 M sodium sulfate (Na2S04) and incubated for 40 min at 37°C in a solution containing 10 mM Tris HCl, 1.1 mM Tris base, 6.7 mM calcium chloride (CaCl& 3.3 mM potassium ferrocyanide [ K4Fe( CN)6], 0.5 mM potassium ferricyanide [K3Fe(CN)s], 1% ethanol, and 0.5 mM 5-bromoindoxyl acetate to elucidate motor end plates, which appear light blue in this reaction. Sections were then dehydrated in 70 and 100% ethanol before being fixed to the slide with 0.5% (wt/vol) celloidin. The secured sections were fixed at room temperature in buffered formal-saline (pH 7.0) containing 3.7% formaldehyde, 14.5 mM sodium chloride (NaCl), 1.3 mM acid sodium monophosphate (NaH,PO& and 1.8 mM anhydrous disodium phosphate (Na,HPO,) and fixed again at 37°C in 0.6 M chloral hydrate before a 40-min incubation at 37°C in a solution containing 1.2 M silver nitrate (AgNO,) and 4 mM cupric sulfate (CuSOJ. Sections were developed in a solution containing 90 mM hydroquinone and 0.4 mM sodium sulfite ( NaS03), fixed in 0.2 M sodium thiosulfate (Na2S203), toned with 5 mM sodium tetrachloroaurate (NaAuC&), and fixed again with 0.2 M Na2S203. MEP and terminal nerve data were quantified in MEPs (n = 1,320) as follows: 1) MEP area, 2) the number of terminal nerves per MEP, 3) the summated lengths of all terminal nerves within a MEP, and 4) the ratio of the total nerve length to the MEP area as an indication of the density of neuronal contact (28). An additional sample of approximately 200 MEPs per muscle was used to quantify the percentage of MEPs that were void of terminal nerves. For the histochemical and MEP data the means derived from individual muscles were averaged, resulting in the mean t SE of the mean. A two-way analysis of variance was used to analyze the data for main effects of age and cross-reinnervation and for interactions between age and experimental treatment. The Bonferroni post
Downloaded from www.physiology.org/journal/ajpcell by ${individualUser.givenNames} ${individualUser.surname} (137.154.019.149) on January 13, 2019.
C98
CROSS-REINNERVATION
OF
SKELETAL
hoc test for critical differences was used to compare individual means, and P 5 0.05 was considered significant.
IN
AGED
RATS
muscles of 12-mo rats was normally distributed over a wide range of fiber areas compared with other groups, with a small percentage of fibers < 1,000 pm2 (Fig. 1). In control EDL muscle there was an increase from 12 to 29 mo in the ratio of total terminal nerve length to MEP area (Fig. 2, A and C, Table 2), which indicates a higher density of neuronal contact at individual neuromuscular junctions in muscles from 29-mo rats. The higher density from 12 to 29 mo of age resulted from a 50% decrease in MEP area, a 23% decrease in terminal nerve number, and an 18% decline in total terminal nerve length (Table 2). Compared with control EDL muscles from 12-mo rats, cross-reinnervation resulted in a 26% increase in the density of neuronal contact caused by a 27% decrease in MEP area and no change in nerve terminal number nor total terminal nerve length (Fig. 2B, Table 2). There were no differences in any of the MEP and terminal nerve parameters between self- and cross-reinnervated muscles in 12-mo rats. Compared with control EDL muscles in 29-mo animals, cross-reinnervation resulted in a 50% decrease in density due to a 48% decrease in terminal nerve number and 44% decrease in total terminal nerve length with no change in MEP area (Fig. 20, Table 2). In 29-mo rats, differences in MEP area between self- and cross-reinnervated muscles accounted for the increased density of neuronal contact in crosscompared with self-reinnervated muscles. The number of noninnervated MEPs increased with age in control EDL muscles (1 vs. 6% in 12 and 29 mo, respectively). After self- and cross-reinnervation, the number of noninnervated MEPs increased compared with control EDL muscle in both l2- and 29-mo rats. The magnitude of the increase was greater in 29-mo rats (17% in 12 mo 1 anu average Jzoer cross-sectzonal area
RESULTS
By 29 mo of age, body mass decreased 23% from the 12mo value of 438 t 7 g (Table 1). There was no difference in body weight due to experimental intervention at either age. Fifty-six days after operations, self-reinnervated EDL muscle mass was 41 and 40% less than the control values of 162 t 3 and 112 t 3 mg for 12- and 29-mo animals, respectively (Table 1). Mass of the cross-reinnervated EDL muscle was 13 and 14% lower than the self-reinnervated value of 98 t 3 and 73 t 3 mg from 12- and 29mo animals, respectively (Table 1). Protein concentration of muscle did not differ due to age but decreased 12 and 16% compared with control EDL muscle in self- and cross-reinnervated muscles, respectively (Table 1). There was no difference in protein concentration between the self- and cross-reinnervated groups. Fiber cross-sectional area (CSA) of self-reinnervated EDL muscles decreased 30% from the control value of 2,490 t 310 pm2 in 12-mo rats and decreased 32% from the control value of 1,866 t 195 pm2 in 29-mo rats (Table 1). The average fiber CSA of cross-reinnervated muscles decreased 44 and 33% compared with the fiber area of self-reinnervated muscles in l2- and 29-mo rats, respectively (Fig. 1, Table 1). However, most of fibers from self- and cross-reinnervated EDL muscles of 29-mo rats were
