Biomarkers of drug-induced vascular injury.

In pre-clinical safety studies, drug-induced vascular injury is an issue of concern because there are no obvious diagnostic markers for pre-clinical or clinical monitoring and there is an intellectual gap in our understanding of the pathogenesis of this lesion. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary targets, smooth muscle and endothelial cells are unknown. However, evaluation of novel markers for potential clinical monitoring with a mechanistic underpinning would add value in risk assessment and management. This mini review focuses on the progress to identify diagnostic markers of drug-induced vascular injury. Von Willebrand factor (vWF), released upon perturbation of endothelial cells, is transiently increased in plasma prior to morphological evidence of damage in dogs or rats treated with vascular toxicants. Therefore, vWF might be a predictive biomarker of vascular injury. However, vWF is not an appropriate biomarker of lesion progression or severity since levels return to baseline values when there is morphological evidence of injury. A potential mechanistically linked biomarker of vascular injury is caveolin-1. Expression of this protein, localized primarily to smooth muscle and endothelial cells, decreases with the onset of vascular damage. Since vascular injury involves multiple mediators and cell types, evaluation of a panel rather than a single biomarker may be more useful in monitoring early and severe progressive vascular injury.

Absence of skeletal muscle lesions after experimental restraint stress induced by pancuronium in normal and stress-susceptible pigs.

Stress-susceptible (SS) pigs develop rhabdomyolysis and increased serum levels of muscle enzymes after a 12 min experimental stress induced by the depolarizing myorelaxant succinylcholine. It is suspected that not only the stress situation but also succinylcholine itself contributes to the skeletal muscle lesions. This experiment was performed to study whether rhabdomyolysis occurs after restraint stress when succinylcholine was replaced by the non-depolarizing myorelaxant pancuronium. Four normal and four SS pigs were subjected to restraint stress by intravenous injection of pancuronium. The neuromuscular block was reversed after 12 min by neostigmine. The animals wee necropsied approximately 48 h after stress and 24 skeletal muscle groups were examined pathologically. The severity of acute myofibre lesions were graded, and the results were compared with the results from normal and SS pigs which had been subjected to restraint stress induced by succinylcholine. The serum levels of creatine kinase (CK) and aspartate aminotransferase (ASAT) stayed at the base line level after the stress. The scores for muscle lesions were significantly lower, both in normal and SS pigs, than after restraint stress induced by succinylcholine indicating no rhabdomyolysis after restraint stress induced by pancuronium. Thus succinylcholine is synergistic with stress, exacerbating its effect on skeletal muscle in SS pigs.

The reduction of skeletal muscle lesions after experimental stress in stress-susceptible pigs protected with dantrolene.

Malignant hyperthermia (MH) is a naturally occurring disease of stress-susceptible (SS) pigs subjected to triggering agents or stress. MH is characterized by accelerated muscle metabolism and hyperthermia due to abnormally increased myoplasmic Ca2+ levels. Dantrolene is used for the treatment of MH and acts by reducing the myoplasmic Ca2+ levels. Muscle lesions can be induced by experimental restraint stress in SS pigs and are suspected to be caused by increased myoplasmic Ca2+ levels. This experiment was performed in order to study if stress induced muscle lesions could be reduced by dantrolene. Nine SS pigs were exposed to experimental restraint stress provoked by a 12 min intravenous (i.v.) infusion of the depolarizing myorelaxant succinylcholine. Five pigs were orally dosed with dantrolene (5 mg/kg), twice, about 24 and 5 h before the stress (group A). The other four pigs were treated with a single i.v. infusion of dantrolene (5 mg/kg), 30 min before stress (group B). The animals were necropsied approximately 48 h after the stress and 24 skeletal muscles were examined macro- and microscopically. No clinical signs of MH occurred during the experiment. The required dose of succinylcholine was higher in group B (0.08 mg/kg/min) than in group A (0.03 mg/kg/min) indicating decreased sensitivity to succinylcholine in SS pigs after i.v. treatment with dantrolene. The pigs in group A, but not in group B showed slightly increased serum levels of creatine kinase (CK) and aspartate aminotransferase (ASAT) at time of necropsy. A significant reduction in acute muscle lesions was observed in both groups, especially in group B, when compared with SS pigs subjected to restraint stress, but not treated with dantrolene. The muscle lesions induced by the stress model are considered to be induced by increased myoplasmic Ca2+ levels since they can be reduced by dantrolene treatment.

Distribution and morphology of skeletal muscle lesions after experimental restraint stress in normal and stress-susceptible pigs.

Six normal and 22 stress-susceptible (SS) pigs were subjected to experimental restraint stress to test the hypothesis that SS pigs are more affected by stress-induced skeletal muscle lesions than normal pigs. The stress was provoked by a 12 min intravenous infusion of the myorelaxant succinylcholine at a dose which induced leg paralysis. At necropsy 2-3 days after the stress, 24 muscles were examined macro- and microscopically. The plasma levels of noradrenaline and adrenaline increased significantly during the stress, especially in SS pigs. Significantly higher scores of acute muscle lesions (degeneration and phagocytosis) were recorded in the SS pigs than in the normal pigs. The antebrachial flexor muscles, m. gastrocnemius, crural flexor muscles, m. serratus and m. intercostalis were most affected, while m. semitendinosus, m. masseter, crural extensor muscles, m. quadriceps and antebrachial extensor muscles were the least affected. The muscle regeneration of SS pigs was greater than that of normal pigs indicating more active rhabdomyolysis in SS pigs than in normal pigs. The muscle lesions were also reflected in increased serum levels of creatine kinase (CK), aspartate aminotransferase (ASAT), potassium and creatinine in SS pigs. It is concluded that the restraint stress induced skeletal muscle lesions and increased sympathetic activity, predominantly in SS pigs. It was also shown that certain skeletal muscles are more affected by rhabdomyolysis than others.

Reduction of heart lesions after experimental restraint stress: a study in stress-susceptible pigs.

Pigs, crossbreeds of Swedish Landrace and Yorkshire, about 6 months old and susceptible to develop malignant hyperthermia when exposed to halothane, were subjected to a 12-min experimental stress provoked by the myorelaxant succinylcholine. The experimental pigs were pre-treated before the stress: five were given propranolol for one week, six were given alpha-tocopherol (vitamin E) combined with selenium for 11 days, and five pigs were pre-treated with zinc (ZnSO4) for 1 month. A total of 12 untreated, stress-susceptible pigs served as controls. The blood levels of noradrenaline and adrenaline recorded during the stress were significantly reduced in the groups pre-treated with propranolol or alpha-tocopherol combined with selenium. The results show significant reduction of myocardial necrosis by beta-adrenoceptor-blocking agents and free-radical scavengers during stress-induced increased sympathetic activity.

Improved biocompatibility of intraocular lenses by heparin surface modification: a 12-month implantation study in monkeys.

To evaluate the long-term biocompatibility and potential side effects of heparin surface modification of a poly(methyl methacrylate) intraocular lens (IOL), a heparin surface modified IOL was implanted in the left posterior chamber of 24 cynomolgus monkeys and a reference IOL (without surface modification) was implanted in the right eye in 12 of these animals. Twelve eyes were not operated on. Eleven eyes in seven monkeys were lens extracted as a control of the surgical method. Slitlamp examinations and intraocular pressure recordings were made one day, one and two weeks, and 1, 2, 2 1/2, 3 1/2, 6, 8, 10, and 12 months after the operation. Eleven monkeys were sacrificed after 3 1/2 months and the remaining animals after 12 months for morphological examination of the eyes. Slitlamp and morphological examinations showed that cell deposits, pigmentation, and posterior synechias were significantly less in eyes with heparin surface modified IOLs than in eyes with reference IOLs throughout the 12-month observation period. The intraocular pressure was equally reduced in eyes with heparin surface modified IOLs and reference IOLs for about one month, after which it returned to normal. No side effects following the implantation of heparin surface modified IOLs were observed. We concluded that heparin surface modification of IOLs is efficient for long-term reduction of cell deposits and posterior synechias after implantation in monkey eyes and may also be effective in lowering the degree of side effects to IOL implantation in humans.

Cardiac manifestation and blood catecholamine levels during succinylcholine-induced stress of malignant hyperthermia sensitive pigs.

Fifteen crossbred pigs of Swedish Landrace and Yorkshire, about 6 months of age and susceptible to develop malignant hyperthermia (MH) when exposed to halothane, were subjected to stress provoked by the myorelaxant succinylcholine. The results were compared with those of 12 normal pigs. During the stress the halothane-sensitive (HS) pigs showed much higher levels of plasma noradrenaline and adrenaline and more severe ventricular arrhythmias than the controls. The degree of myocardial degeneration and necrosis being similar to catecholamine induced myocardial damage was significantly higher in the HS pigs than in the controls. The ultrastructural examination revealed three main types of changes in affected myocardial cells. One type of myocardial cell damage was characterized by various degree of hypercontraction, enlarged mitochondria with dense bodies and dilated sarcoplasmic reticulum. The other type showed mitochondria with tubular configuration whereas the third type of cell damage was characterized by almost normal mitochondria combined with a severe damage of the myofilaments. Three HS pigs which died within 30 min after stress showed signs of malignant hyperthermia. No signs of the disease were observed in the other 12 HS pigs.

Disordered catecholamine release in pigs susceptible to malignant hyperthermia.

Pigs, crossbreeds of Swedish Landrace and Yorkshire, females and castrated males about 6 months old, were exposed to experimental stress. The pigs were either considered normal or shown to be susceptible to develop malignant hyperthermia when tested with halothane at about 6 weeks of age (stress-susceptible pigs). The stress was of the restraint type, produced by two different myorelaxant agents, the depolarizing succinylcholine or the non-depolarizing pancuronium. The blood levels of the catecholamines (CA) noradrenaline (NA) and adrenaline (A) were measured during the stress. The severity of myocardial cell necrosis observed 1 to 2 days after the stress was morphologically graded. In normal pigs the levels of NA during the stress and the degree of myocardial cell necrosis were about the same after both succinylcholine and pancuronium. In stress-susceptible pigs, however, succinylcholine produced very high NA and A levels and severe heart lesions, whereas after pancuronium the NA and A levels were rather low and the heart lesions significantly reduced when compared to those after succinylcholine-induced stress. After pretreatment with dantrolene intravenously the succinylcholine-induced stress only induced slightly increased blood CA levels and no signs of myocardial cell necrosis in pigs susceptible to develop malignant hyperthermia. Dantrolene, an efficient drug in treatment of malignant hyperthermia, probably acts by interfering with release of calcium from the sarcoplasmic reticulum in skeletal muscles. The results indicate that peripheral sympathetic neurones in MHS pigs also react abnormally, probably due to defective calcium turn-over.

Catecholamine-induced free radicals in myocardial cell necrosis on experimental stress in pigs.

Normal pigs, crossbreeds of Swedish Landrace and Yorkshire, about 6 months old, were subjected to experimental stress, induced by the myorelaxant succinylcholine, for 12 min. Besides one group of control pigs, one group of pigs were pretreated with alpha-tocopherol (vitamin E) combined with selenium (Tokosel Vet) injected i.m. every second day for 11 days, and another group was given zinc (ZnSO4.7H2O) in the fodder for 1 month plus one injection i.p. 2 days before the stress. The stress-induced heart lesions, morphologically graded according to evaluation scores, were significantly reduced in both the pretreated groups when compared with the control pigs. The blood levels of catecholamines (CA) were increased to about the same degree in the three groups during the stress. The protection observed is suggested to be due to the fact that vitamin E, selenium and zinc are involved in systems acting as scavengers of free radicals. The present results together with earlier ones are discussed to support the CA-hypothesis for stress-induced heart lesions: some types of stress can increase the sympathetic activity to such an extent that released CA, via beta-adrenoceptor mechanisms affects the cell metabolism to such a degree that cytotoxic free radicals are formed, producing myocardial cell necrosis.

A histopathological study of the inner ear after the administration of hyaluronan into the middle ear of the guinea pig.

Hyaluronan, 19 mg/ml, was administered into the middle ear cavity of guinea pigs. A control group was subjected to a sham operation. Fourteen days after administration the animals were killed and a macroscopic examination of all middle ears was performed. Surface preparations of the organ of Corti were made and examined by light microscopy. Hair cell loss along the length of the sensory epithelium was quantified. No toxic effect of hyaluronan could be detected either in the middle ear or in the sensory cells in the cochlea. Thus hyaluronan is potentially useful for facilitating middle ear surgery.

An ultrastructural and functional study of the inner ear after administration of hyaluronan into the middle ear of the guinea pig.

A single dose of hyaluronan, 19 mg/ml was administered into the right middle ear cavity of guinea pigs. The auditory function of the right ear was tested by recording the gross neural action potential (N1) before, directly after, and 28 days after the administration of hyaluronan. In a control group the right ear was sham-operated and the gross neural action potential (N1) was recorded twice; on the day of the sham operation and 28 days later. All animals were killed 28 days after the operation. All treated as well as sham-operated ears and every second intact ear were studied by scanning electron microscopy (SEM.) No ear had signs of hearing deterioration. Macroscopically, most of the hyaluronan was eliminated from the middle ear after 28 days. In the histological examination, pathological stereocilia were found in the apical turn of the cochlea in the ears to which hyaluronan was administered, the sham operated and the intact ears.