ARTICLE |
Correspondence to: L. De-Doncker, Laboratoire de Plasticité Neuromusculaire, EA 1032, IFR 118, Bât. SN4, Université des Sciences et Technologies de Lille 1, 59655 Villeneuve d'Ascq Cedex, France. E-mail: neuromus@pop.univ-lille1.fr
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Summary |
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Morphological, contractile, histochemical, and electrophoretical characteristics of slow postural muscles are altered after hindlimb unloading (HU). However, very few data on intrafusal fibers (IFs) are available. Our aim was to determine the effects of 14 days of hindlimb unloading on the morphological and immunohistochemical characteristics of IF in rat soleus muscle. Thirty-three control and 32 unloaded spindles were analyzed. The number and distribution of muscle spindles did not appear to be affected after unloading. There was no significant difference in number, cross-sectional area, and histochemical properties of IF between the two groups. However, after unloading, a significant decrease in slow type 1 MHC isoform and a slight increase in slow-tonic MHC expression were observed in the B and C regions of the bag1 fibers. The -cardiac MHC expression was significantly decreased along the entire length of the bag2 fibers and in the B and C regions of the bag1 fibers. In 12 muscle spindles, the chain fibers expressed the slow type 1 and
-cardiac MHC isoforms over a short distance of the A region, although these isoforms are not normally expressed. The most striking finding of the study was the relative resistance of muscle spindles to perturbation induced by HU. (J Histochem Cytochem 50:15431553, 2002)
Key Words: rat, soleus, hindlimb unloading, intrafusal fibers, histochemistry, myosin heavy chain isoforms
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Introduction |
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THE MUSCLE SPINDLES are stretch receptors that inform the central nervous system about changes in rate and muscle length (-cardiac, slow-tonic, and fast-twitch). Nuclear bag1 fibers express four MHC isoforms (embryonic, slow-twitch, slow-tonic, and
-cardiac) and nuclear chain fibers express two MHC isoforms (neonatal and fast-twitch). The importance of the primary endings to normal spindle development has been repeatedly demonstrated by selectively eliminating the sensory or motor supply in fetal or neonatal rats (for review see
-motor neurons. It is known that the Ia afferents not only innervate IFs but also project onto
-skeletomotor and ß-skeletofusimotor neurons, and onto interneurons which, in turn, project on to
-fusimotor neurons in the spinal cord (
-fusimotor neurons that innervate the contractile portion of the IF. Therefore, the aim of this study was to identify the MHC isoform distribution along IFs and to determine if this distribution was modified by unloading. A panel of eight different antibodies, in combination with ATPase labeling, was used.
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Materials and Methods |
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Animals
Eight male Wistar rats (Iffa Credo; l'Arbresle, France) weighing 280300 g were randomly divided into two groups of four rats: control rats and HU rats. Initially, all of the rats were housed in the same room at a constant temperature (25C) with a 12-hr:12-hr lightdark cycle and were fed and allowed water ad libitum. Animals in the HU group were unloaded for 14 days using Morey's model (
Tissue Processing
Rats were anesthetized with sodium pentobarbital (35 mg·kg-1). In both groups, the right and left soleus muscles were excised, stretched to their resting length, and immediately frozen in an isopentane solution precooled to its freezing point by liquid nitrogen. The muscles were stored at -80C until histochemical and immunohistochemical analyses were performed. The soleus muscles of the control and HU groups were cut into serial frozen transverse sections (10 µm thick) using a cryostat microtome (Leica CM 1800; Heidelberg, Germany) set at -20C. Along the muscle, every 280 µm, 11 sections perpendicular to the muscle longitudinal axis were performed. The first section was used as a negative control in the immunohistochemical analysis. The next two sections were processed for myofibrillar adenosine 5'-triphosphatase (ATPase) with acid (pH 4.3) and alkaline (pH 10.4) preincubations according to the method of
Antibodies and Labeling
Eight monoclonal antibodies (MAbs) were used in this study (Table 1). Binding of the primary antibodies was localized by an immunoperoxidase reaction utilizing the Novostain Universal Quick Kit (Tebu-Novocastra; Le PerrayenYvelines, France). Serial cross-sections were incubated in prediluted blocking serum (normal horse serum) for about 10 min. The excess serum was blotted and sections were incubated with primary antibodies diluted in PBS for 2 hr. Serial cross-sections were washed for 5 min in PBS. Then the sections were incubated for 30 min in prediluted biotinylated universal secondary antibody. At the end of 30 min, sections were washed with PBS for 5 min and incubated in ready-to-use streptavidinperoxidase complex reagent for 30 min. To label the serial cross-sections, the peroxidase substrate solution (diaminobenzidine, DAB) was added after the sections had been washed for 5 min with PBS. Positive fibers were characterized by a brown color and negative fibers remained unlabeled. Finally, after dehydration with alcohol and toluene, the slides were mounted in Eukitt resin. The cross-sectional area (CSA) and the densitometry of the antibody labeling along the different IF types were measured using an image analyzer (SAMBA 2005; Villeneuve d'Ascq, France). To avoid a disparity in the labeling, for each antibody, all the sections were identically processed with the same diluted solution. The slides were also incubated simultaneously for 5 min in a common DAB solution. The zero of the densitometer for each preparation was adjusted on the negative fibers in the section; the slight variations of labeling between each slide were therefore discarded. The immunohistochemically labeled slides were analyzed with the image analyzer. This system was made up of a digital camera fixed to a standard optical light microscope. The camera was coupled to a computer with video monitor and image acquisition and storage modules. The measures were quantified to 256 gray levels (level 1 corresponding to black color and to 0% of light transmission, and level 256 to white color and 100% of light transmission), which were then converted automatically into optical density, taking into account the studied area. The densitometric values were expressed in percentage (OD = log10 (1/light transmission). According to
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Identification of IF Types
The muscle spindles were identified in both control and unloaded soleus muscles groups as encapsulations of small-diameter fibers. As stated previously, each muscle spindle can be divided into two encapsular regions (A and B regions) and one extracapsular portion (C region). Intrafusal fibers were classified as nuclear bag1, nuclear bag2, and nuclear chain fibers according to their morphology and their histochemical reaction after preincubation in acid and alkaline solutions (-cardiac MHC isoform. Nuclear chain fibers did not express these two MHC isoforms, except in a very short equatorial length where the slow-tonic isoform was detected (
Statistical Analysis
All results were expressed as means ± SD. A Student's t-test was used to establish the intergroup comparisons and statistical significance was accepted at p<0.05.
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Results |
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A total of 33 control muscle spindles (33 bag2, 33 bag1, 69 chain fibers) and 32 HU muscle spindles (32 bag2, 32 bag1, 67 chain fibers) were analyzed throughout their entire length. Most of the muscle spindles in the control and unloaded groups contained one bag1, one bag2, and two chain fibers. However, in three control and four HU muscle spindles, there were three nuclear chain fibers. There was no significant different between the number of muscle spindles in control (14.3 ± 1.5) and HU groups (13.5 ± 1.3).
Cross-sectional Areas
The CSAs of IFs are shown in Fig 1. For both control and HU groups, the CSAs of bag2 fibers were significantly larger than those of bag1 and chain fibers, and those of bag1 fibers were higher than those of chain fibers, independent of the muscle spindle region. For all IF types in both animal groups, the CSAs in the B region were significantly higher than those in the A and C regions (Fig 1). After 14 days of unloading, no significant difference in the CSAs of all IF types between control and HU groups was observed.
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Histochemical Analysis
In Fig 2 (control group), the Figures 2.12.3 and Figures 2.42.6 respectively illustrative the acid (pH 4.3) and alkaline (pH 10.4) ATPase activity of IFs in the A, B, and C regions of a muscle spindle. In the muscle spindles of the control group, the nuclear bag1 fibers had low to moderate acid ATPase activity in A and B regions, but the activity was high at the end of the B region and towards the poles. After alkaline preincubation, they showed a low ATPase activity along their entire length. Nuclear bag2 fibers exhibited high acid ATPase activity and high to moderate alkali ATPase labeling along their length. However, in the more extracapsular region, the nuclear bag2 fibers lost their alkaline ATPase activity. In general, after acid preincubation, nuclear chain fibers exhibited low ATPase activity and high to moderate alkali ATPase labeling along their entire length. However, the nuclear chain fibers in the muscle spindles of Fig 2 exhibited moderate acid ATPase activity in the A region. In the most equatorial region, all IF types had a rim or no ATPase activity. Histochemical labeling did not show any difference between control and HU groups. However, it is obvious that a slight increase or decrease in one of the MHC isoforms would not necessarily be made visible by changes in ATPase activity. Moreover, ATPase labeling does not allow demonstration of the co-expression of several MHC isoforms within a fiber.
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Immunohistochemistry
The regional variation of labeling with all antibodies is illustrated in Fig 2 for the control group. Fig 3 illustrates only the antibody labelings that were changed by the HU condition. The results have been refined by densitometric measurements (Table 2, control group and Table 3, HU group).
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Control Group Nuclear Bag1 Fibers. NCL MHCs (Figures 2.72.9) labeled only the fibers towards the poles at the end of the B region and in the C region. SC71 (Figures 2.102.12) and ALD 58 MAbs (Figures 2.162.18) strongly labeled the fibers in A and B regions and the labeling slightly decreased towards the C region (Table 2). The nuclear bag1 fibers were labeled with F88 MAb (Figures 2.192.21) over a short distance in the B and C regions. The binding with F88 MAb was strong to moderate in the B region and low to moderate towards the C region. The labeling with 2B6 MAb (Figures. 2.252.27), specific for embryonic MHC, was low in the A region. No binding was seen in the B and C regions of bag1 fibers. Whatever the IF MY32 (Figures 2.132.15), BFF3 (Figures 2.222.24), NCL MHCd MAbs did not label the nuclear bag1 fibers (not shown).
Nuclear Bag2 Fibers. NCL MHCs MAb reacted strongly with the nuclear bag2 fibers along their entire length. The nuclear bag2 fibers bound MY32 and F88 MAbs along their entire length but the binding intensity decreased from the A to the C region (Table 2). With SC71, ALD58, and 2B6 MAbs, the nuclear bag2 fibers were labeled only in the A region. BF-F3 MAb reacted in both A and B regions of bag2 fibers; the labeling was lower in the B region. NCL MHCd MAb did not label bag2 fibers.
Nuclear Chain Fibers. Nuclear chain fibers reacted with MY32 MAb in the A and B regions. BF-F3 labeled the nuclear chain fibers in the A and B regions, with higher labeling in the B region (Table 2). SC71 MAb bound only the A region of nuclear chain fibers. Our results showed no labeling with NCL MHCs, ALD58, F88, 2B6, and NCL MHCd MAbs in any region of the nuclear chain fibers.
HU Group. There was no difference with the control group in the regional variation and in the labeling intensity with SC71, MY32, BF-F3, 2B6, and NCL MHCd MAbs along the nuclear bag1 and bag2 fibers.
Nuclear Bag1 Fibers.
NCL MHCs, ALD58, and F88 MAbs (Figures 3.43.6, 3.103.12, and 3.163.18, respectively) showed modified labeling compared to the control group (Figures 3.13.3, 3.73.9, and 3.133.15, respectively). Densitometric analysis, presented in Table 3, showed that type 1 MHC expression in nuclear bag1 fibers decreased significantly in both B and C regions after unloading. Moreover, the labeling intensity with ALD58 MAb, specific for slow-tonic MHC isoform, was significantly increased in the B and C regions of bag1 fibers. With F88 MAb, our results showed that the expression of -cardiac MHC isoforms in bag1 fibers was significantly decreased in the B and C regions after unloading.
Nuclear Bag2 Fibers. After unloading, the labeling intensity with F88 MAb significantly decreased in all regions of the nuclear bag2 fibers compared with the control group. There was no difference with the control group in the regional variation and the labeling intensity with ALD58 MAb along bag2 fibers. However, although the difference was not significant, type 1 MHC isoform expression presented a slight decrease in the B and C regions of the nuclear bag2 fibers after a period of unloading.
Nuclear Chain Fibers. There was no difference with the control group in the regional variation and labeling intensity along the nuclear chain fibers with all antibodies, except for NCL MHCs and F88 MAbs. Indeed, among the 32 muscle spindles that were studied, only 12 presented nuclear chain fibers that bound the NCL MHCs and F88 MAbs (Figures 3.4 and 3.16, respectively) over a short distance of the A region (special chain in Table 3), whereas chain fibers in the control group never bound these antibodies.
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Discussion |
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Morphological Characteristics
The number and distribution of muscle spindles in the rat soleus were not significantly different in the two experimental groups. These data are in agreement with those described previously by
Histochemical Analysis
In the control group, our data are similar to those obtained by other authors for the same species (
After a 14-day period of HU, no significant difference was observed in the ATPase labeling in the IFs compared to the control group. The regional variation in ATPase labeling along the IFs is due to the non-uniform expression of MHC isoforms in the different regions of those fibers (
MHC Isoform Expression of IFs in Control and HU Groups
Control Group.
Our results are in agreement with other studies concerning the regional variation of labeling with NCL MHCs (
However, some differences were observed with SC71, BF-F3, 2B6, and NCL MHCd antibodies. Our results showed that SC71 bound to the encapsulated polar region of bag1 fibers and labeling was also seen in the juxtaequatorial region in bag1 and chain fibers. Our results were in accordance with those described by
A difference in labeling was observed in the nuclear bag1 fibers with MY32 and SC71 MAbs. Nuclear bag1 fibers were labeled by SC71 along their entire length, whereas MY32 MAb never labeled these fibers. This was surprising because the MY32 MAb is supposed to react with all fast-twitch MHC isoforms (
Our results show that the BF-F3 MAb bound only the nuclear bag2 and chain fibers in the A and B regions, whereas
Only a low level of labeling or no labeling was observed with 2B6 and NCL MHCd antibodies, respectively. These labeling differences reflected either differences in antibody affinities or the existence of more than one embryonic MHC isoform (
HU Group.
All the observations on regional variation and labeling intensity in muscle spindles of the control group were also valid for the HU group. However, after unloading, some differences were observed in the expression level of slow type I, slow-tonic, and -cardiac MHC isoforms.
The regulation of the -cardiac MHC isoform expression along the length of bag fibers is under the influence of motor innervation. The expression of
-cardiac MHC appears one day after the arrival of bag1 and bag2
-motor innervation (
-cardiac MHC isoform was decreased and was limited to a shorter portion of the fibers, whereas the nuclear bag1 fibers were unreactive (
In adult rat muscle spindles, deafferentation or de-efferentation produced less severe alterations, but the expression pattern of some MHC isoform along the IF was modified (-cardiac MHC in the outer B region, ceased to express this isoform. Moreover, bag2 fibers continued to express this isoform but less intensely than normally.
-cardiac MHC was decreased and the slow-tonic expression was increased along the length of nuclear bag fibers. According to these studies, we suggest that the decrease in
-cardiac and the increase in slow-tonic MHC isoforms in nuclear bag fibers reflect a decrease in the activity pattern of the motor nerves during unloading. This hypothesis is reinforced by the fact that, when extrafusal fibers are deprived of motor innervation (
It is more difficult to understand why slow-twitch and -cardiac MHC isoforms are expressed over a very short distance of the A region in nuclear chain fibers after unloading. The expression of these two MHC isoforms is usually dependent on motor innervation, whose influence decreases from the C to the A region. Therefore, the expression of slow-twitch and
-cardiac MHC isoforms over only a short portion of the A region was unexpected. No previous studies on fetal, neonatal, or adult muscle spindles have reported a possible afferent or motor influence on the expression of slow-twitch and
-cardiac MHC isoforms in nuclear chain fibers.
To conclude, after unloading, the muscle spindle integrity and the labeling pattern of the majority of MHC isoforms were preserved. However, some differences were observed: a decrease in -cardiac MHC expression and an increase in slow-tonic expression along the nuclear bag fibers. In the literature, it has been demonstrated that the level of
-cardiac MHC expression (
-skeletomotor neurons but also onto ß-skeletofusimotor and
-fusimotor neurons (
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Acknowledgments |
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Supported by grants from the CNES (3027) and the Conseil Régional du Nord Pas-De-Calais.
The SC-71 and BF-F3 antibodies developed by
Received for publication January 18, 2002; accepted May 29, 2002.
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