Figure 1 shows serial sections of rat gastrocnemius after 1 hour of treadmill running. The VEGF mRNA signal is apparent as a dark stain (panel A), and may be compared to the sense control (panel B) which shows no non-specific binding in the same muscle region. In panel C, type I (dark stain), IIa (light stain), and IIb-IId/x (intermediate stain) fibers can be identified based on the myofibrillar actomyosin ATPase histochemical staining procedure 18. Immunohistochemistry was used to positively identify subpopulations of fibers expressing only the 2B MHC. Panel D shows positive binding (dark) of BF-F3 antibody against 2B MHC. In this manner subpopulations of IIb and IId/x fibers were identified. Panel E shows positive binding (dark) of the NCL-MHCs antibody against the slow MHC (type I), and panel F shows positive binding (dark) of the A4.74 antibody against 2A MHC. Identification of I and IIa fibers by immunohistochemistry was fully concordant with identification of these fiber types by the myofibrillar actomyosin ATPase histochemical staining procedure.

Figure 2 shows representative Northern blots for total VEGF mRNA levels examined in (A) resting control, (B) exercised, and (C) electrically stimulated muscles. It is clear that VEGF mRNA levels are elevated over resting after both 1 hour of treadmill exercise and 1 hour of electrical stimulation. This is shown clearly in Figure 3 which gives the quantitative densitometry for VEGF mRNA, normalized to 18S ribosomal RNA. A significant increase in VEGF mRNA over resting control is observed after 1 hour of treadmill running (~3.5-fold, P = 0.015), and after 1 hour of electrical stimulation (~3.5-fold, P = 0.015). There were no significant differences in the VEGF mRNA levels between exercised and electrically stimulated muscles.

Oxidative regions were located in the deep part of the gastrocnemius, and were identified based on the expression of a majority of type I and IIa fibers (~77% of the total fiber number in the regions analyzed). Glycolytic regions were located superficially, and were identified based on the expression of only type IIb and IId/x fibers.

The heterogeneity of fiber phenotypes in oxidative regions affords the possibility of comparing VEGF mRNA signal between adjacent fibers differing in metabolic capacity. However, data collected in this regard were considered insufficient to test the hypothesis that fiber phenotype is related to VEGF mRNA signal strength. That is, a non-biased test of this hypothesis requires a random sampling of fibers from each phenotypic class in multiple muscle samples. Such fibers must then be clearly identified on serial slide preparations to determine fiber phenotype and VEGF mRNA signal. This was not possible in all muscle samples due to freezing artifact and tissue degradation during (particularly) the in situ hybridization process. Regional comparisons are not limited in this way as muscle regions were easily identified on serial sections for measurement of VEGF signal in a random sample of fibers within the region (see Methods).

Data presented in Table 1 show the normalized mean VEGF mRNA signal in oxidative versus glycolytic muscle regions from rats after 1 hour of treadmill exercise, rats after 1 hour of electrical stimulation, and control rat muscle (neither exercise or electrical stimulation). Mean VEGF signal was determined from 27 randomly sampled fibers in each defined region within a single muscle sample (see Methods). By ANOVA, there were significant differences in VEGF mRNA signal strength between functional regions (P = 0.002) and between protocols (treadmill, electrical stimulation, and control, P = 0.036). In treadmill run muscle the glycolytic regional mean was 93% of the oxidative regional mean (P ≤ 0.05), and in electrically stimulated muscle the glycolytic regional mean was 59% of the oxidative regional mean (P ≤ 0.05). Significant regional differences were not apparent in control muscle (P = 0.463). Post-hoc testing reveals a significantly larger regional difference in VEGF mRNA signal in electrically stimulated versus treadmill run muscle (P = 0.017), and no significant difference in this regard between electrically stimulated and control muscle, although the latter comparison reaches a P-value of 0.056. Regional differences were not significantly different between treadmill run and control muscle (P = 0.912).

This study was approved by the University of California, San Diego, Animal Subjects Committee. Female Wistar rats were used (age 6–8 weeks, ~200 grams body weight). All animals were housed in cages and allowed standard rat food and water ad libitum prior to study.

Both treadmill exercise and muscle electrical stimulation protocols were used to initiate the total VEGF mRNA response in rat skeletal muscle. For the exercise protocol, 6 rats were first familiarized with a rodent treadmill (Omnipacer model LC-4, Omnitech, Columbus, OH), and then required to run for 1 hour at 20–35 m/min on an incline of 10°. Running speed varied between rats but was maintained for an individual rat near the maximal running speed that could be sustained for a 1 hour period. For rats of this age and body size we have previously shown that a running speed of 20 m/min at 10° incline represents ~55% of maximal oxygen consumption 10.

For the electrical stimulation protocol 6 rats were anesthetized by i.p. injection of sodium pentobarbitol. Because anesthetization depresses ventilatory drive, and because hypoxia can also induce VEGF mRNA upregulation, great care was taken in the electrical stimulation protocol to ensure that animals were adequately mechanically ventilated. For each rat, the trachea was intubated and ventilation was maintained at a tidal volume of 2.5 ml, ventilatory frequency of 50 breaths/min, and 1 cm of positive end expiratory pressure. Preliminary work showed this level of ventilation to be sufficient to preserve normal arterial PO₂ and PCO₂. The left gastrocnemius, soleus, and plantaris muscles were surgically isolated and the sciatic nerve, which innervates this muscle complex, was cut and attached to an electrode. The hindlimb was fixed so that no movement of the limb was possible during muscle contraction and the Achilles tendon was attached to a force-displacement transducer (Grass Instruments, Co., Quincy, Mass) to monitor tension development during contraction. Contractions were initiated by train rate impulses, 4–8 volts, 200-ms duration, 1 ms^-1 frequency, given at a rate of 2 stimulations (contractions) per second for 1 hour. Signs of muscle fatigue i.e., a decline in tension development below 75% of peak, were evident using this stimulation protocol after 30–60 minutes in approximately 50% of the rats tested. In the remaining rats, tension was well maintained over the course of the stimulation protocol. There was no detectable difference in VEGF mRNA levels by Northern analysis between muscles that fatigued versus muscles that maintained tension development. Six resting rats (non-exercised) were processed as a control for Northern Analyses and in situ hybridization. These were the age matched cage mates of those rats run on the treadmill (2 rats per cage). With the exception of the exercise protocol, these rats were handled in an identical manner to the exercised rats.

After the exercise protocol rats were allowed to rest for 1 hour and then were anesthetized by i.p. injection of sodium pentobarbitol. For the 6 rats run on the treadmill, the left gastrocnemius (both heads combined) was removed for RNA isolation, frozen in liquid nitrogen, and stored at -80°C. In these same rats the right gastrocnemius was removed for in situ hybridization and fiber type determinations. This tissue was mounted in TBS tissue freezing medium (Triangle Biomedical Sciences, Durham, NC), frozen in isopentane cooled in liquid nitrogen, and stored at -80°C. For the 6 electrically stimulated rats, skeletal muscle was also harvested 1 hour after the completion of the electrical stimulation protocol. Electrical stimulation can be applied to one leg at a time only. In our protocol the left leg is used. Thus, the left gastrocnemius was harvested for RNA isolation and Northern analyses in six rats. An additional four rats provided a left gastrocnemius for in situ hybridization, histochemistry, and immunohistochemistry. This minor inconsistency in study design is not expected to affect study results. Six resting rats provided a right gastrocnemius as a control for Northern analysis.

For Northern analysis, 18 muscle samples were initially processed (6 rest control, 6 electrically stimulated, and 6 treadmill run), but some samples were degraded during the RNA isolation leaving 3 rest-control, 4 exercised, and 4 electrically stimulated muscle samples. In order to ascertain regional and fiber type patterns of VEGF mRNA production in those muscles stimulated to upregulate VEGF, 4 each of treadmill exercised and electrically stimulated muscle samples, and 2 control muscles, were successfully processed through in situ hybridization.

Total cellular RNA was isolated from each gastrocnemius muscle by the method of Chomczynski and Sacchi 32. RNA preparations were quantified by absorbance at 260 nm and RNA intactness and integrity assessed by ethidium bromide staining after separation by electrophoresis in a 6.6% formaldehyde-1% agarose gel. Fractionated RNA was transferred by Northern blot to Zeta-probe membrane (Bio-Rad, Hercules, CA). Following transfer, RNA was cross-linked to the membrane by ultraviolet irradiation for 1 min and stored at 4°C. The blots were then probed with oligolabeled [alpha-³²P] deoxycytidine triphosphate cDNA probes which had a specific activity > 1 × 10⁹ disintegrations.min-1.μgDNA-1(11). The rat VEGF probe is a 0.9 kb cDNA Pst I/Sma I insert cloned into a pBluescript II KS+ vector 33. Prehybridization and hybridization were performed in 50% formamide, 5×SSC (20×SSC is 0.3 M sodium chloride, 0.3 < sodium citrate), 10× Denhardt's solution (50× Denhardt's solution is 2% Ficoll, 2% polyvinyl pyrrolidone, 2% Bovine Serum Albumin Factor V), 50 mM sodium phophate (pH 7.0), 1% sodium dodecyl sulfate (SDS), and 250 μg/ml salmon sperm DNA at 42°C. Blots were washed with 2×SSC and 0.1% SDS at room temperature and 0.1% SSC and 0.1% SDS at 65°C. Blots were exposed to XAR-5 X-ray film (Eastman Kodak, New Haven, CT) for 2–3 days by use of a Cronex Lighting Plus screen at -80°C. Autoradiographs were quantitated by densitometry within the linear range of signals and normalized to ribosomal 18S RNA levels.

Frozen gastrocnemius muscle samples were cryo-sectioned in a Reichert Jung Cryocut 1800 cryostat (Cambridge Instruments, Buffalo, NY) at -20°C to 10 μm and mounted in cross-section on Fisherbrand Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Slides were immediately processed through the in situ hybridization protocol of Braissant and Wahli 20 to detect VEGF mRNA transcripts on tissue sections. After tissue fixation for 20 minutes in 4% paraformaldehyde in diethyl pyrocarbonate (DEPC) treated phosphate buffered saline (PBS), sections were incubed for 2 × 15 minutes in PBS containing 0.1% active DEPC, and equilibrated for 15 min in 5×SSC. The sections were then prehybridized for 2 hr at 58°C in the hybridization mix (50% formamide, 5×SSC, salmon sperm DNA 40 μg/μl; 500 μl on each section). The hybridization probes (see below) were denatured for 5 min at 80°C and added to the hybridization mix (400 ng/ml). The hybridization reaction was carried out overnight at 58°C with 50 μl of hybridization mix on each section. During hybridization, sections were kept in a box saturated with 5×SSC, were covered by a rectangle of Parafilm, and were sealed with DPX Mountant (Fluka Chemie, Neu-Ulm, Switzerland) to prevent drying. After hybridization, sections were washed for 30 min in 2×SSC (room temperature), 1 hour in 2×SSC (65°C), 1 hr in 0.1×SSC (65°C), and equilibrated for 5 min in Buffer 1 (Tris-HCl 100 mM and NaCl 150 mM, pH 7.5). Sections were then incubated for 2 hours at room temperature with alkaline phosphatase-coupled anti-digoxigenin antibody (Boehringer Mannheim, Indianapolis, IN) diluted 1:5000 in Buffer 1 containing 0.5% Blocking reagent (Boehringer Mannheim). Excess antibody was removed by two 15 min washes in Buffer 1, and the sections were equilibrated for 5 min in Buffer 2 (Tris-HCl 100 mM, NaCl 100 mM, and MgCl₂ 50 mM, pH 9.5). Color development was performed at room temperature overnight in Buffer 2 containing NBT and BCIP (Boehringer Mannheim). Staining was stopped by a 10 min wash in Tris/EDTA (10/1 mM, ph 8.0), and non-specific staining was removed by 1 hour in 95% EtOH. Sections were rehydrated in deionized water for 15 min to remove precipitated Tris and then dehydrated through successive ethanol baths (70, 95, 100%) and Hemo-De (Fisher Scientific) (2 × 15 min each) and mounted in Permount (Fisher Scientific).

Hybridization probes were derived from a ~600-bp cDNA that contains the entire coding region of the human VEGF₁₆₅8 subcloned into the pBluescript II KS+ vector (Stratagene, La Jolla, CA). The final riboprobe was the same size (~600-bp) as the cDNA. Constructs in this vector were linearized at appropriate restriction sites to allow the sythesis of digoxigenin-UTP labeled complementary RNA in the antisense or sense orientation (using T3 or T7 RNA polymerase, respectively; Boehringer, Indianapolis, IN). The riboprobes synthesized in the "sense" orientation served as background control.

Fiber types were determined in serial cross sections. Two methods were used: (1) The histochemical method of Ogilvie and Feeback 18, based on staining for myofibrillar actomyosin ATPase, and (2) immunohistochemistry based on the reaction of specific antibodies for mysosin heavy chain (MHC) isoforms 3435. In the histochemical staining procedure, sections were pre-incubated in (0.025 M potassium acetate, 9 mM calcium chloride-dihydrate, pH to 4.5 with glacial acetic acid) for 8 minutes, rinsed 3× two min/each in (0.1 M Trizma base (Sigma), 0.018 M calcium chloride-dihdrate), and incubated for 25 minutes in (0.154 M ATP-disodium salt, 0.053 M Glycine, 0.029 M calcium chloride, 0.065 M sodium chloride, 0.048 M sodium hydroxide, pH 9.4). Following incubation sections were rinsed 3× in 0.014 M calcium chloride-dihydrate, stained for 1 min in Toluidine blue, rinsed for 5 sec in deionized water, and dehydrated with 5 dips in 95% ethanol, 5 dips in 2 changes of 100% ethanol, and 2 changes of Hemo-De (Fisher) for 5 min each. Sections were mounted with Permount (Fisher). This method allows for the clear determination of type I and IIa fibers, but distinguishing IIb from IId/x subpopulations is difficult and subjective as both stain with nearly the same intensity. For this reason immunohistochemistry was also used on one muscle sample to clearly identify IIb fibers based on specificity of the 2B MHC antibody. For immunohistochemistry, mouse polyclonal and monoclonal antibodies specific for slow (Novocastra, NCL-MHCs), 2A (Blau, A4.74), and 2B (DSM, BF-F3) MHC isoforms were used. Serial sections were incubated overnight with primary antibodies diluted (NCL-MHCs, 1:100; and BF-F3, A4.74, 1:1) in a 0.2 M potassium phosphate solution (PPS). After washing 3× with PPS, sections were incubated for 3 hr at room temperature with either peroxidase-conjugated goat anti-mouse immunoglobins (for NCL-MHCs, and A4.74) or biotinylated rabbit antimouse IgM (for BF-F3) (DAKO A/S, Glostrup, Denmark). The latter was also incubated for 1 hour with streptavidin-HRP (NEN Life Science Products, Boston, MA) diluted 1:100 in PPS. Sections were again washed 3× in PPS and color was evolved by incubating slides in the dark between 2–10 min in a 3,3'-diaminobenzidine (DAB) solution (Vector Laboratories Inc., Burlingame, CA). Sections were rinsed in deionized water, dehydrated in successive ethanol baths, cleared with Hemo-De (Fisher), and mounted with Permount (Fisher).

Regional VEGF mRNA signal within an individual muscle fiber was quantified by densitometry from microscope images obtained by a Sony 3ccd color video camera (Sony Corporation, Japan) attached to a Jenalumar optical microscope (Jenoptik JENA, Germany), and processed by the Sigma Scan Pro imaging software (SPSS Inc., Chicago, IL). Optical density measurements were made on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available from the internet by anonymous FTP from http://zippy.nimh.nih.gov). This program can calculate mean grey-scale value within the cross sectional area of a single muscle fiber.

Unfortunately, measured grey-scale values give arbitrary units. Within a given microscopic field of view, an optical density measurement depends both on the strength of the VEGF mRNA signal, and also on the amount of light transmitted through the tissue sample (which in turn depends on the degree of freezing artifact in muscle preparation and many other factors). Uncontrolled factors affecting light transmission imply that optical density comparisons between different slide preparations are not valid. In order to aggregate data from different slides, it was necessary to normalize all VEGF mRNA optical density signals to an internal standard. For example, to describe the difference between an oxidative and glycolytic muscle region, the optical density value of each individual fiber was normalized to the mean of all fibers within the oxidative region (which was arbitrarily set to a value of 1.0) (see Table 1).

A random sampling strategy was used to obtain mean optical density values for different muscle regions identified on histochemically stained sections. Oxidative regions were located in the deep part of the gastrocnemius and expressed a majority of type I and IIa fibers (~77%), while glycolytic regions were located superficially, and expressed only type IIb and IId/x fibers. For each muscle, a microscopic field of view was located in a region which contained typically between 100–200 fibers in cross section at 125× magnification. The same field of view was then located on a serial section processed through in situ hybridization. Twenty seven fibers total (three fibers from each of 9 equal quadrats) were randomly selected from the region on the in situ slide preparation and densitometric measurements of the VEGF mRNA signal within fibers were made.

Analysis of variance was used to test for differences in VEGF mRNA levels from Northern analysis between muscle samples from exercised (n = 4) and electrically stimulated rats (n = 4). ANOVA, using the individual muscle sample as the unit of analysis, was also used to test for the main effects of functional region (oxidative vs. glycolytic) and protocol (treadmill running, electrical stimulation, control) on VEGF mRNA signal. Post-hoc testing, using Fisher's Least Square Difference correction for multiple comparisons was used to test for differences between protocols in the regional difference of VEGF mRNA signal. Paired t-tests were used to compare regional means within a given protocol. All statistical analyses were performed using Systat Version 5.2 (Systat Inc., Evanston, IL). Statistical significance was set at p ≤ 0.05 for all tests.