Chapter Twenty-Two: Non-MH Research

Chapter 22 Sub-sections

Barbiturate Tolerance

Research ideas sometimes apparently came out of nowhere. Part of our care for patients undergoing neurosurgery involved prevention or treatment of diminished blood flow --- ischemia --- to areas of the brain, with the possible problem of a stroke. Barbiturates such as thiopental (Sodium Pentothal®) were being used for these purposes because they suppressed metabolism in the brain and constricted blood vessels, theoretically improving the chances for success. But, with greater doses and prolonged use, their therapeutic effect waned, due to tolerance to the drug. So we investigated this in dogs, examining responses of the whole body as well as individual organs.

Barbiturates were valuable in cerebral protection during brain ischemia, but what were the limitations? Tolerance might mitigate protection, and there could be unexpected specific organ damage with time. Tolerance could be related to enzyme induction of metabolism, where metabolism increases due to enzyme stimulation, or to receptor changes.

With the latter, greater blood concentrations are needed for a similar effect. We did not observe this. We examined multi-organ system responses via Theye's approach: isolate organ venous drainage and use the Fick equation to determine individual organ metabolism (Theye et al, 1975). Dogs developed tolerance within the brain within three hours, manifested as an increase in cerebral oxygen consumption with constant blood levels of pentobarbital (Gronert, Michenfelder et al, 1981). Similar findings were noted in other systems: whole body, gastrocnemius muscle, kidney, splanchnic region, and heart, without evidence for toxicity (Gronert, Michenfelder et al, 1983).

It was surprising, and remains unexplained, that tolerance occurred as a change in metabolism and not as a need for greater blood concentrations of depressant. In addition, there was no apparent toxicity of barbiturates when used in these large doses, and for at least three hours. We confirmed what Theye had postulated: changes in whole body oxygen consumption during general anesthesia are due to the sum of events in individual organs and tissues, and an anesthetic-induced change in function correlates with a change in metabolic requirements (Theye et al, 1975). These metabolic events were observed when tolerance developed, where the change in requirement is not due to an increase in function (Gronert, Michenfelder et al, 1981, 1983).

Upregulation of AChRs --- Downregulation of AChRs
Study of Muscle Disuse Atrophy

Back to our considerations of disuse activity: from our early studies, we knew that burn patients had an agonist sensitivity, observed as an exaggerated potassium efflux after receiving succinylcholine (Tolmie et al, 1967; Schaner et al, 1969; Gronert, Dotin et al, 1969), a response also observed in patients with denervated muscle (Birch et al, 1969; Cooperman et al, 1970; Case History, 1971; Gronert, 1970). We wondered why inactive muscle not directly involved in burns would also have altered responses to a muscle relaxant.

Since all burn patients suffer muscle disuse atrophy from confinement to bed, and great weight loss, we believed that disuse might be a vital factor. Our canine studies of immobilization disuse atrophy ruled out disuse as the sole factor because the extra potassium efflux after succinylcholine was only slightly greater than normal, about 10% of the quantitated denervated response (Gronert, Lambert et al, 1973; Gronert, Theye, 1975).

But then another milestone was reached: resistance to non-depolarizing muscle relaxants was reported in patients with upper motor neuron lesions (Graham, 1980; Moorthy et al, 1980) and in burn patients (Martyn et al, 1980). These confirmed the earlier observation of resistance to curare noted years earlier (Bush, 1964). It could not be an unrelated coincidence that these disparate lesions imposed an exaggerated response, i.e., sensitivity to succinylcholine, and a diminished response, resistance, to non-depolarizing relaxants. We therefore examined qualitatively the response of canine gastrocnemius muscle suffering immobilization disuse atrophy to the non-depolarizing skeletal muscle relaxant pancuronium, and observed resistance (Gronert, 1981). This resistance was confirmed with a more sophisticated quantitative study (Gronert, Matteo et al, 1984). On a hunch, because exercise is the opposite of disuse, we examined metocurine pharmacodynamics in exercise conditioned dogs, and observed sensitivity (Gronert, White et al, 1989). This all began prior to our awareness of receptor theory and up- and downregulation of nicotinic acetylcholine receptors (Kenakin et al, 1992).

My reading began to clarify this area (Kenakin, 1989), and this, in time, resulted in an article on up- and downregulation of skeletal muscle nicotinic acetylcholine receptors with various disorders (Martyn et al, 1992).

Anti-epileptic Drugs and Resistance to Non-depolarizing Relaxants

In addition to skeletal muscle disuse atrophy, resistance to non-depolarizing muscle relaxants occurs in patients treated with anticonvulsants, drugs used to treat convulsive disorders, or epilepsy. These act in multi-synaptic areas to control seizures and have a spillover effect at the neuromuscular junction, a facet of their structure-activity relationship in blocking synaptic transmission (Alderice et al, 1980). This attenuation of the effect of acetylcholine results in a weak non-depolarizing blockade, of which patients seem unaware.

When the anticonvulsant is first started, this weak blockade potentiates the potency of non-depolarizers. If surgery is needed during this period, non-depolarizers or curare type drugs would be needed in lesser amounts. After about two weeks, this weak blockade results in proliferation, or upregulation of acetylcholine receptors, with resistance to non-depolarizers, manifested in part as a shorter duration of action (Messick et al, 1982). In further research, as a manifestation of this upregulation of receptors during anticonvulsant therapy, we observed the typical accompanying sensitivity to a cholinergic agonist, e.g., succinylcholine, as prolonged action (Melton et al, 1993). Newer anti-convulsant drugs do not seem to have this property (Audu et al, 2006). All these considerations led to a complex study of sedated dogs.

Canine Intensive care: three weeks, 24 hours/day, Sedated Ventilated

I had wondered for some years about the effects of muscle disuse atrophy, skeletal muscle that is forced to rest, and never contract, but with no lesion that could cause direct damage, e.g., denervation or other abnormality. I'd performed some studies regarding this, but still had puzzles: How much alteration is there in responses of muscle that is without activity, but no other problems? I followed this for some years, beginning with my years at the burn unit in San Antonio, for burn patients had peculiar alterations in muscle function despite having only a burn injury on the skin. I had superficially pursued the literature regarding disuse, beginning early with a monograph from a 1962 symposium (Gutmann et al, 1963). We had over the years performed several studies of casted leg immobilization but wished to expand to a study of leg muscle disuse, or total inactivity, with no physically restricting immobilization. My experience at an NIH meeting expanded my interest and this led to this next study.

Background: FDA Meeting in Bethesda

The FDA Advisory Committee Meeting on Anesthetic and Life Support Drugs convinced me that additional study of ICU-related immobilization was warranted (FDA Advisory, 1992). At that meeting, we discussed problems in ICU patient weakness (FDA Advisory, 1992: Summary, Appendix #5). On page 2 of Appendix #5, Drs. Miller (Chair, UCSF), Roizen (Chair, U Chicago), Hoyt (U Pittsburgh), and Gronert discuss the difficulties in evaluating ICU complications and some concluded that in depth prolonged ICU studies would be impossible. This ‘impossible' conclusion bothered me, and I mulled it over during the trip back to UC Davis.

I decided to study dogs in an intensive care unit with collaborators from the UC Davis medical and veterinary schools. This is a hugely difficult study, for it requires skilled monitoring, 24 hours a day, for 21 straight days. A variety of experiences had prepared our laboratory for planning and arranging this study of extended prolonged animal care. My past studies had included porcine thermal trauma, with debridement and cleansing twice per day during a six week period, and anesthesia once per week (Fox et al, 1947; Gronert, Theye, 1971), tolerance in dogs during a 24 hour period of pentobarbital anesthesia (Gronert, Michenfelder et al, 1981; 1983), and close observation of 48 hour studies by Michenfelder et al of cerebral ischemia plus five day follow-up (Michenfelder et al, 1976).

All of these prior studies involved cooperation, organization, a well equipped laboratory, planning, and reliable skilled technicians. We fortunately had skilled colleagues at UC Davis: veterinary anesthesiologists with ICU experience, and experienced capable full time technicians in our laboratory. When I returned to UCD, I discussed these factors with two colleagues, and we formulated a protocol.

The Study Itself

Previously healthy active dogs, two or three at a time, were heavily sedated, and their tracheas intubated, with mechanical ventilation. This labor intensive study required direct skilled personal care 24 hours per day for the three weeks of the study, with immediate presence or immediate backup by faculty for problems. The worst problem was a power failure in the middle of the night, handled magnificently. The lone veterinary student managed to ventilate both dogs via reservoir bags, and simultaneously call the emergency phone number (911) for aid. Both dogs survived uneventfully.

Strict careful handling and intensive unit care was maintained for three weeks, for fluids, gastric feedings, change of position, temperature control, and bowel and bladder function (Gronert, Haskins et al, 1998). Each week, all dogs were challenged with a brief infusion of the non-depolarizing relaxant metocurine, with measurement of degree of paralysis, time, and plasma levels of metocurine.

Figure 20
Fig. 20. Exaggerated resistance to metocurine in dogs with sedation-related muscle disuse atrophy. Normal canine IC50 is about 250 ng/ml.

Pharmacokinetic and pharmacodynamic analysis (Shafer et al, 1989; Fung et al, 1995) estimated the degree of resistance to metocurine with time, and thereby estimated the degree of upregulation from total disuse atrophy due to heavy sedation without exposure to skeletal muscle relaxants.

These dogs showed greater upregulation with disuse atrophy than previously observed, e.g., resistance to the test non-depolarizing relaxant, metocurine, Fig. 20 (Fig 1, in Gronert GA, Fung DL, Haskins SC, Steffey EP, Deep sedation and mechanical ventilation without paralysis for 3 weeks in normal beagles, Anesthesiology 1999, June, number 6, 90:1741-5, copyright owner Lippincott, Williams & Wilkins,, used with permission.) This confirmed our suspicion that a total loss of volitional muscle activity would greatly exaggerate the increase in potency of metocurine that we had reported during immobilization disuse atrophy. With casted immobilization, volitional movement is still possible, and likely, within the cast.

Relationship of Exercise and Graded Muscle Disuse

Because we had used the same protocol involving the skeletal muscle relaxant metocurine in a series of canine studies for some years, and our approaches regarding drugs and study analyses were similar, we compared the results. Chronic conditioning exercise resulted in sensitivity to metocurine (smaller doses

Figure 21
Fig. 21. Comparison of sensitivity to metocurine with varying degrees of exercise or disuse atrophy

than normal produced the same degree of paralysis) and graded amounts of disuse resulted in graded degrees of resistance to metocurine. Thus there is a gradation, depending upon the primary stress upon muscle, and presumably related to numbers of functioning acetylcholine receptors. Fig. 21. (Fig 2, in Gronert GA, Fung DL, Haskins SC, Steffey EP, Deep sedation and mechanical ventilation without paralysis for 3 weeks in normal beagles, Anesthesiology 1999, June, number 6, 90:1741-5, copyright owner Lippincott, Williams & Wilkins,, used with permission.) The figure showing our results summarizes some relationships regarding skeletal muscle function in several situations: exaggerated use such as conditioning exercise, normal use, modest disuse atrophy from cast immobilization, and total muscle disuse with intense sedation.

Our canine extreme example of ICU sedation and marked paralysis mirrors the human situation in which very ill ICU patients have been given large doses of non-depolarizers to aid in management of their ventilation. These patients would also have increased numbers of functioning acetylcholine receptors, and cause them to potentially react to succinylcholine with dangerous hyperkalemia. Case reports support this conclusion (Bolton CF, 2005; Martyn et al, 2006). All these data support Kenakin's statements (1989; 1992) that the skeletal muscle motor nerve-endplate represents a classic competitive agonist/antagonist system. Acetylcholine and non-depolarizing muscle relaxants compete at the endplate receptor. When there are more acetylcholine molecules (the agonist) than relaxant molecules (the competitive antagonist), the muscle contracts when a nerve impulse arrives. When there are more relaxant molecules, acetylcholine released by the nerve impulse cannot reach the endplate receptor, and there is paralysis.

Ironically, while two of my colleagues participated in planning the ICU protocols, neither were co-authors on the published material. One withdrew in the early stages, due to his own research projects. The other had participated in depth, but disagreed on an approach that involved two separate papers. I felt that we needed a basic article on "how to" as regards the challenging animal care and its success, and a clinical paper describing the exaggerated upregulation demonstrated by the muscle relaxant metocurine.

He declined co-authorship on both papers; he did not complain after the first paper appeared, but did so vigorously after the second. We exchanged letters concerning this conflict with the journal editor and the UC Davis Administrative Offices. He and I disagree as to precisely how this all happened (Letters, Beagle study, 1999). This unfortunate interaction rigorously reinforced my awareness that planning should be confirmed in writing, signed by all.

Species with Unique Muscle Receptor Responses

An interesting species is the pronghorn (antelope), an astounding athlete. It can continuously run 40 miles per hour or so, for relatively long distances (Lindstedt et al, 1991). With this degree of exercise conditioning, would the antelope have greater sensitivity to a non-depolarizing relaxant, i.e., be down-regulated, similar to our exercised dogs (Gronert, White et al, 1989; Martyn et al, 1992)? We had several pronghorns for potential study via the veterinary physiologist Jim Jones at UC Davis, but problems developed, and we could not pursue their relaxant studies.

Another example is the mongoose. It is resistant to poisoning by α-bungarotoxin of snake venom, yet it is a mammal with apparently normal functioning skeletal muscle nicotinic acetylcholine receptors (Keller et al, 1995). How different is the blocking effect of a non-depolarizing relaxant in the mongoose from the various mammals we studied? What might that hint at regarding structure-activity relationships?

Unfortunately, despite continued attempts, we could not secure any for study. The response may be instructive in determining structure-function relationships in a variety of situations of varying activity and innately altered receptor responses.