Newton

DAMAGE AND REPAIR

The bupivacaine protocol for local muscle damage and repair was employed in rat

skeletal muscle to determine the mechanisms by which the dietary supplements whey

protein and creatine may be exerting their effects. Two animal studies were designed to

replicate the human experimental design and supplementation protocol. Study three

examined the effects of creatine supplementation on the metabolic aspects of

chemically-induced muscle damage in rat skeletal muscle (i.e. the calcium handling

ability of the muscle), and subsequent damage and repair processors, while the fourth

study examined the effects of whey protein on the regeneration (recovery) aspect of

chemically-induced muscle damage.

A total of sixty-four male Sprague-Dawley rats (Rattus norvegicus) weighing 212.4 +

3.5 (+ SEM) were randomly separated into 3 groups: i) control n= 20; ii) creatine-

supplemented n=23; and iii) whey protein-supplemented n=21. A total of 9 rats were

not included in thesis analysis. 8 rats were directly affected by problems with the

heating bath of the contractile testing apparatus, while 1 rat died of anesthetic overdose

preceding contractile force testing. Following the same format as the human studies, the

experimental design and dietary supplementation protocol for study three and four will

be discussed in detail in subsequent chapters. Hence, only the procedures used in both

studies will be discussed in this chapter.

3.2.1 Experimental Injury

All rats were lightly anesthetized with a domitor (10 mg.kg-1 body weight,

Medetomidine Hydrochloride, Novartis Animal Health, NSW, Australia) and ketamine

Australia) combination via an intraperitoneal injection (i.p.), such that they were

unresponsive to tactile stimuli. The extensor digitorum longus (EDL) and soleus (SOL)

muscles of the left hindlimb were surgically exposed, with care taken to avoid

damaging the nerve and blood supplies. A total of ~600µl of 0.5% bupivacaine

(Bupivacaine Hydrochloride, Pharmacia and Upjoin (Perth) Pty. Ltd, WA, Australia)

was injected into both EDL and soleus by two injections in each of the proximal,

midbelly, and distal regions of the muscle using a 26 gauge needle (Terumo, USA) (see

figure 3.5). This procedure ensured the degeneration of most fibres in the injected

muscle (Rosenblatt, 1992). Previous research has shown that injection of Bupivacaine

remains contained within the targeted muscle, leaving both nearby blood and nerve

supplies intact (Louboutin et al., 1996). This model of injury is specific for muscle

fibres (Rosenblatt and Wood, 1992), thereby eliminating the complicating factors of

damage to motor nerves and associated blood vessels that occur with other models of

muscle injury (Best et al., 2000). The contralateral EDL and soleus muscle served as an

uninjured control and was not injected intramuscularly, as previous research has shown

that little or no damage is caused by the insertion of the needle itself, or the injection of

an otherwise harmless vehicle such as isotonic saline (Rosenblatt, 1992).

Figure 3.5 Muscles of the left lower hindlimb (taken from Greene, 1963). EDL (green) and soleus (red).

Following injury, the small incision was closed by Michel clips (Fine Science Tools,

Nth Vancouver B.C, Canada), and swabbed with Betadine antiseptic (povidone iodine

solution, Faulding Pharmaceuticals, SA, Australia). Previous research has shown that in

this type of minor operative procedure, michel clips are a preferable option to sutures

(Gregorevic et al., 2000). Following the surgical procedure, anesthesia was reversed

using Antisedan (125ug.kg-1 body weight, Atipamezole Hydrochloride, Novartis

Animal Health , Australasia Pty. Ltd, NSW, Australia) and animals either continued

their dietary modification or were randomly separated into a control or whey protein-

supplemented group.

At 7 and 14 days post-injury, rats received intra-peritoneal injections of 1 ml.kg-1 body

weight Nembutal (10 mg.kg-1 body weight, Rhone Merieux, QLD, Australia). The level

of anaesthesia was monitored via corneal and footpad reflexes, and additional 0.1ml

Nembutal injections applied when required. A longitudinal incision was then made into

the injured hind leg of the rat. The EDL (predominantly type II fibres) was excised first,

and fully tested (Section 3.3.1) before the same dissection procedure as described below

was performed on the soleus (predominately type I fibres). The aim of fully testing the

EDL muscle while other muscles remained intact in the anaesthetized animal, was to

preserve the integrity of these muscles by maintaining blood and nerve supply for the 1-

2 hours of experimental testing of each muscle.

3.2.2 Dissection Protocol

The proximal tendon of the EDL is located first and surrounding tissue and muscle was

cleared away. Surgical silk size 2.0 (LA-55G Ethicon, Johnson & Johnson, NSW,

Australia) was tied securely at the point of proximal tendon insertion of the EDL

tendon insertion. The muscle was carefully dissected free from other tissues, starting

from the proximal end, and placed into a horizontal custom-built plexiglass bath (see

figure 3.6 and 3.7) containing a Krebs Henseleit Ringer solution [NaCl 118mM; KCL

4.75mM; Na2HPO4 1mM; MgSO4.7H2O 1.18mM; NaHCO3 24.8mM; CaCl2 2.5mM

and D-Glucose 11.0mM]. This HCO3- based buffer solution was maintained at a pH of

7.4, by being aerated with carbogen (95% O2 and 5% CO2) (BOC gases, Melbourne,

Australia). All experiments were conducted at 25oC as isolated muscles do not remain

viable for long periods of time if tested at 37oC. These experimental conditions facilitate

optimal oxygen diffusion throughout the muscle, and maintain stability of functional

measurements in vitro (Ryall et al., 2002).