Age-dependent intoxication by larkspur (Delphinium) in Angus steers.

The effect of age on larkspur poisoning of cattle is unknown. An experiment consisting of oral dosing of dried, ground, Delphinium barbeyi to ten Angus steers as yearlings, and again at two years was performed. There was a significant difference between the responses of yearling and two year old steers (P = 0.0015), with yearling steers being more susceptible. These results suggest that the adverse response of Angus cattle to larkspur is age-dependent.

Complete inhibition of fetal movement in the day 40 pregnant goat model by the piperidine alkaloid anabasine but not related alkaloids.

Four chemically similar alkaloids, anabasine, anabaseine, epibatidine and dimethylphenylpiperazinium (DMPP), are potent nicotinic acetylcholine receptor agonists of fetal muscle nicotinic acetylcholine receptors in human TE-671 cells. Based on results with these cells, we hypothesized that the alkaloids would completely inhibit ultrasound-monitored fetal movement in a goat model. Different, single doses of anabasine, anabaseine, epibatidine, DMPP, or saline control were administered I.V. to pregnant goats on day 40 of gestation and the number of fetal movements per 5 min sample was measured by ultrasound at times 0, 0.5, 1, 2, 4 and 8 h. The differences among does in fetal movements were more consistent at dosing and following recovery for doses of anabasine above 0.125 mg/kg compared to the other compounds and dosages. Anabasine actions were dose-dependent with an IC50 value of ∼0.1 mg/kg, and, at a dose of 0.8 mg/kg, completely inhibited fetal movement for 1.5 h after dosing. Anabaseine, epibatidine, and DMPP failed to completely inhibit fetal movement in day 40 pregnant goats at doses predicted to be effective. These results suggest that while experiments with TE-671 cells provide valuable information and predictions of the actions of plant alkaloids on fetal movement, in vivo experiments are still required in order to determine the ability of an alkaloid to inhibit fetal movement in livestock species. Moreover, other pharmacological properties such as receptor differences between mammalian species and differences in the pharmacokinetic properties of the alkaloids also are likely to weaken teratologic predictions based solely on the in vitro data.

Serum toxicokinetics after intravenous and oral dosing of larkspur toxins in goats.

Poisoning of cattle by larkspur plants (Delphinium spp.) is a concern for cattle ranchers in western North America. Previous research studies have evaluated the toxicokinetic profile of multiple larkspur toxins in several livestock species. However, those studies were all performed by orally dosing plant material. Consequently some toxicokinetic parameters could not be definitively determined. In this study, we compared the serum toxicokinetic profile of the larkspur alkaloids methyllycaconitine (MLA) and deltaline in goats dosed both IV and via oral gavage. The results from this study indicate that the toxic alkaloids in larkspurs undergo flip-flop kinetics, meaning the rate of absorption of the alkaloids is slower than the rate of elimination. The implications of flip-flop kinetics in treating animals poisoned by larkspur is discussed.

Administering multiple doses of a non N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-containing tall larkspur (Delphinium occidentale) to cattle.

Larkspurs (Delphinium spp.) are a serious toxic plant problem for cattle in western North America. There are two chemotypes of D. occidentale, a more toxic and a less toxic chemotype. The objective of this study was to evaluate the acute toxicity of the less toxic chemotype when administered in multiple doses to cattle. These results suggest that cattle could consume enough of the less toxic chemotype to be poisoned in a range setting.

Comparison of the serum toxicokinetics of larkspur toxins in cattle, sheep and goats.

Larkspurs (Delphinium spp.) are a major cause of cattle losses in western North America, whereas sheep are thought to be resistant to larkspur toxicosis. Goats are often used as a small ruminant model to study poisonous plants. In this study, we compared the serum toxicokinetic profile of toxic larkspur alkaloids from Delphinium barbeyi in cattle, goats, and sheep. The results from this study indicate that kinetic differences could partially explain species differences in susceptibility to larkspur toxicosis.

White snakeroot poisoning in goats: Variations in toxicity with different plant chemotypes.

Tremetone and possibly other benzofuran ketones are believed to be the toxic compounds in white snakeroot. However, disease has not been reproduced with purified toxins and the concentrations of the benzofuran ketones in white snakeroot populations that cause toxicosis have not been documented. The objectives of this study were to compare the toxicity of seven plant populations, better characterize the clinical and pathologic changes of poisoning, and correlate intoxication with benzofuran ketone content. Four of the seven white snakeroot collections were toxic at the dose and duration used in the study. Affected goats became exercise intolerant, had significant serum enzyme changes and histological lesions in the large appendicular muscles. The incidence and severity of poisoning was not correlated with total doses of tremetone or total benzofuran ketone concentrations suggesting they may not be closely involved in producing toxicity and the possible involvement of an unidentified toxin. The results also demonstrate that white snakeroot populations vary chemically and toxicologically.

The effect of administering multiple doses of tall larkspur (Delphinium barbeyi) to cattle.

Larkspurs ( spp.) are one of the most serious toxic plant problems on foothill and mountain rangelands in the western United States. A considerable amount of research has been conducted over the years in both field and pen settings. The results of these research efforts have significantly increased our understanding of the poisoning of cattle by larkspurs. However, most of the pen studies conducted thus far have used a dosing regimen of a single bolus dose, which does not accurately mimic the manner by which cattle are poisoned by larkspur while grazing. Consequently, the objective of this study was to evaluate the acute toxicity of tall larkspur ( collected near Manti, UT) when administered in multiple doses, with the intent to identify a no observable adverse effect level (NOAEL). The adverse effect selected for this study was muscle weakness to the point the cattle could no longer remain ambulatory as would be required in a grazing environment, thus becoming sternally recumbent when exercised. Hereford steers were administered various doses of tall larkspur at 12-h intervals for 4 d or until they showed marked signs of muscle weakness. The results suggest that a dose of 2 mg kg∙d -(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids is the NOAEL for a tall larkspur population with a norditerpenoid alkaloid profile containing 4 mg MSAL-type alkaloids/g plant material and 12 mg non-MSAL-type alkaloids/g plant material. Additionally, a computer model was generated to simulate multiple-dosing regimens at the various doses as well as different dosing regimens. The results from this study suggest that a 500-kg steer can consume a daily dose of 1.25 kg of fresh tall larkspur (with a similar alkaloid profile) without becoming severely poisoned (suffering from muscle weakness to the point of recumbency). Additionally, these results indicate that a serum concentration of approximately 355 ng methyllycaconitine/mL may represent a toxic threshold.

Studies on the teratogenicity of anabasine in a rat model.

A number of plant toxins have been shown to be teratogenic to livestock. The teratogenic action of some of these alkaloids is mediated by nicotinic acetylcholine receptors (nAChR). However, for many of these alkaloids it is difficult to obtain sufficient quantities of individual alkaloids to perform teratology studies in livestock species. Therefore the objective of this study was to determine if a rat model can be utilized to characterize the teratogenic nature of individual plant toxins that are nAChR agonists. In this study, we evaluated the teratogenicity of anabasine by feeding pregnant rats anabasine-containing rodent chow from gestational day (GD) 6-21. On GD21, the dams were euthanized and the gravid uteri were removed. The gravid uteri and individual pups were weighed. The pups were evaluated for bone malformations including cleft palate and scoliosis. Overall, the results of this study suggest that the rat is not a good model to study the teratogenicity of plant toxins that are nAChR agonists. It is possible that in the rat model, anabasine administered orally via the chow may not result in sufficient reduction in fetal movement to cause the significant malformations observed in livestock species.

Evaluation of the respiratory elimination kinetics of selenate and Se-methylselenocysteine after oral administration in lambs.

Sheep can be acutely poisoned by selenium (Se) accumulating forages which often contain selenate or Se-methylselenocysteine as their predominant forms. Excess Se can be eliminated via respiration. Sheep were given a single oral dose of 0, 1, 2, 3, 4, or 6 mg Se/kg BW as sodium selenate and Se-methylselenocysteine or 6 mg Se/kg BW as sodium selenite or selenomethionine. Expired air samples were collected and analyzed for Se. The Se concentration of the expired air reflected a dose-dependent increase at individual time points for both Se-methylselenocysteine and sodium selenate, however, Se content was greater and eliminated more rapidly from sheep receiving Se-methylselenocysteine. The mean Se concentration in respired air from sheep administered 6 mg Se/kg BW of different selenocompounds was greatest in sheep dosed Se-methylselenocysteine > selenomethionine > sodium selenate > sodium selenite. The Se concentration in respired air of acutely poisoned sheep is significantly different for different chemical forms of Se.

The effect of low larkspur (Delphinium spp.) co-administration on the acute toxicity of death camas (Zigadenus spp.) in sheep.

In most cases where livestock are poisoned by plants in a range setting, there is more than one potential poisonous plant in the same area. Two poisonous plants that are often found growing simultaneously in the same location are death camas (Zigadenus spp.) and low larkspur (Delphinium spp.). Sheep are known to be susceptible to death camas poisoning while they are thought to be resistant to larkspur. The objective of this study was to determine if co-administration of low larkspur would exacerbate the toxicity of death camas in sheep. A dose finding study was performed to find a dose of death camas that caused minimal clinical signs of poisoning. Sheep were observed for clinical signs of poisoning as well as changes in heart rate and muscle fatigue. Sheep dosed with 1.14 g of death camas per kg BW showed slight frothing and lethargy, whereas sheep dosed with death camas and low larkspur showed slightly more noticeable clinical signs of poisoning. Sheep dosed with only low larkspur, at 7.8 g/kg BW, showed no signs of poisoning. Although we observed a qualitative difference in clinical signs of intoxication in sheep co-treated with death camas and low larkspur we did not detect any quantitative differences in heart rate, exercise-induced muscle fatigue, or differences in serum zygacine kinetics. Consequently, the results from this study suggest that low larkspur does not affect the toxicity of death camas in sheep. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas and low larkspur in sheep. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to other livestock species.

Experimental rayless goldenrod (Isocoma pluriflora) toxicosis in horses.

Rayless goldenrod (Isocoma pluriflora) sporadically poisons horses and other livestock in the southwestern United States. Similar to livestock poisoning by white snakeroot (Ageratina altissima) in the midwestern United States, previous research suggests that benzofuran ketones (BFK: tremetone, dehydrotremetone, 6-hydroxytremetone, and 3-oxyangeloyl-tremetone) are responsible for the toxicity of rayless goldenrod. However, experimental reproduction of rayless goldenrod-induced disease and detailed descriptions of poisoning in horses with known concentrations of tremetone and other BFK has not been documented. In this study four horses were fed increasing amounts of rayless goldenrod to obtain doses of approximately 0, 10, 30, and 60 mg BFK/kg BW for 14 days. After seven days of dosing the horse dosed with 60 mg BFK/kg BW horse developed depression, reluctance to eat, dehydration, trembling, and muscle fatigue. Biochemical alterations including increases in the serum enzyme activities of CK, AST, ALT, and LDH, and increased cardiac troponin I concentration, were also identified. Physiologically the clinically poisoned horse had decreased endurance seen as reluctance to perform on the treadmill with increased resting heart rate and a prolonged recovery of heart rate following treadmill exercise. The condition of the horse continued to decline and it was euthanized and necropsied on day 10. At necropsy the myocardium was pale and soft and many of the appendicular and large apical muscles were pale and moist. Histologically, the myocardium had extensive myocardial degeneration and necrosis with extensive fibrosis and multifocal mineralization. Several of the large appendicular muscles in this horse also had small foci of skeletal muscle degeneration and necrosis. Less severe myocardial changes were also identified in the horse dosed with 30 mg BFK/kg BW after 14 days of dosing. No clinical, biochemical or histologic changes were identified in the control horse and the horse dosed with 10 mg BFK/kg BW. These results suggest that doses of 60 mg BFK/kg BW for seven days produce extensive myocardial lesions in horses. The horse dosed with 30 mg BFK/kg BW developed less severe, but similar myocardial lesions over a longer duration, this suggests that poisoning may be cumulative and lower doses of longer duration are also toxic. Horses seem to be uniquely sensitive to rayless goldenrod-induced myocardial disease, therefore cardiac troponin I may be a useful marker of rayless goldenrod poisoning in horses. More work is needed to determine which BFK produce myocardial toxicity and better determine the effects of dose and duration on poisoning in horses.

The role of the α7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllycaconitine and anabasine.

The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. However, recent research demonstrated that there was no difference in the acute lethality (LD50 ) to MLA in mice lacking the α7 nAChR subunit compared with wild-type mice. The objective of this study was to determine if the α7 nAChR subunit plays a role in motor coordination deficiencies that result from exposure to nAChR antagonists and agonists. We compared the motor function and coordination in wild-type mice to mice lacking the α7 subunit of the nAChR, after treating them with a non-lethal dose of MLA or anabasine, using the following tests: balance beam, grip strength, rotarod, open field and tremor monitor. Analysis of the data indicated that overall there was no difference between the wild-type and knockout mice (P = 0.39 for grip strength; P = 0.21 for rotarod; P = 0.41 for balance beam; P = 0.22 for open field; and P = 0.62 for tremors). Thus results from this study suggest that α7 nAChR does not play an integral role in the acute effects of MLA or anabasine on motor function/coordination. Consequently other subunits of nAChRs found in the neuromuscular junction are likely the primary target for MLA and anabasine resulting in motor coordination deficiencies and acute toxicosis.

The role of the α7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research suggested a correlation between the lethal dose (LD50 ) of MLA and the amount of α7 nAChR in various mouse strains, suggesting that mice with more α7 nAChR require more MLA to be poisoned. The objective of this study was to characterize the role of the α7 subunit in the acute toxicosis of MLA by evaluating the acute toxicity of MLA in mice lacking the α7 subunit. The LD50 values for MLA were 4.2 ± 0.9, 3.7 ± 1.1 and 3.3 ± 0.9 mg kg(-1) body weight (BW) for wild-type, heterozygous knockout and homozygous knockout mice, respectively. We also evaluated the response of anabasine in these mice. The LD50 values for anabasine were 1.6 ± 0.3, 2.0 ± 0.4 and 1.8 ± 0.3 mg kg(-1) BW for wild-type, heterozygous knockout and homozygous knockout mice, respectively. The protein expresson of various nAChR subunits was compared to determine if mice lacking the α7 subunit compensate by over expressing other nAChR subunits. There were no significant differences in the protein expression of the α3 , α4 , α5 , ß2 and ß4 subunits amongst the three genotypes of mice in brain or skeletal muscle. The results of this study suggest that α7 nAChR does not play an integral role in the acute toxicosis of MLA or anabasine. Consequently other nAChR subunits of nAChRs found in the neuromuscular junction are probably the primary target for MLA and anabasine resulting in acute toxicosis.

The effect of 7, 8-methylenedioxylycoctonine-type diterpenoid alkaloids on the toxicity of tall larkspur (Delphinium spp.) in cattle.

Delphinium spp. contain numerous norditerpenoid alkaloids which are structurally delineated as 7, 8-methylenedioxylycoctonine (MDL) and N-(methylsuccinimido) anthranoyllycoctonine (MSAL)-type alkaloids. The toxicity of many tall larkspur species has been primarily attributed to their increased concentration of MSAL-type alkaloids, such as methyllycaconitine (MLA), which are typically 20 times more toxic than MDL-type alkaloids. However, the less toxic MDL-type alkaloids are often more abundant than MSAL-type alkaloids in most Delphinium barbeyi and Delphinium occidentale populations. Previous research demonstrated that MDL-type alkaloids increase the acute toxicity of MSAL-type alkaloids. In this study, we examined the role of MDL-type alkaloids on the overall toxicity of tall larkspur plants to cattle while controlling for the exact dose of MSAL-type alkaloids. Cattle were dosed with plant material from 2 different populations of tall larkspur containing either almost exclusively MDL- or MSAL-type alkaloids. These 2 plant populations were combined to create mixtures with ratios of 0.3:1, 1:1, 5:1, and 10:1 MDL- to MSAL-type alkaloids. The dose that elicited similar clinical signs of poisoning in mice and cattle was determined for each mixture on the basis of the MSAL-type alkaloid content. As the ratio of MDL- to MSAL-type alkaloids increased, the amount of MSAL-type alkaloids required to elicit clinical signs decreased. These results indicate that the less toxic MDL-type alkaloids in tall larkspur exacerbate the toxicity of the MSAL-type alkaloids. Consequently, both the amount of MSAL-type alkaloids and the amount of total alkaloids should be fully characterized to determine more accurately the relative toxicity of tall larkspur plant material.

The acute toxicity of the death camas (Zigadenus species) alkaloid zygacine in mice, including the effect of methyllycaconitine coadministration on zygacine toxicity.

Death camas (Zigadenus spp.) is a common poisonous plant on foothill rangelands in western North America. The steroidal alkaloid zygacine is believed to be the primary toxic component in death camas. Poisonings on rangelands generally occur in the spring when death camas is abundant, whereas other more desirable forage species are limited in availability. In most cases where livestock are poisoned by plants in a range setting, there is more than one potential poisonous plant in that area. One common poisonous plant that is often found growing simultaneously in the same area as death camas is low larkspur (Delphinium nuttallianum). Consequently, the objectives of this study were to conduct acute toxicity studies in mice and to determine if coadministration of low larkspur will exacerbate the toxicity of death camas. We first characterized the acute toxicity of zygacine in mice. The LD(50) of zygacine administered intravenously (i.v.) and orally was 2.0 ± 0.2 and 132 ± 21 mg/kg, respectively. The rate of elimination of zygacine from whole blood was determined to be 0.06 ± 0.01/min, which corresponds to an elimination half-life of 13.0 ± 2.7 min. The i.v. LD(50) of total alkaloid extracts from a Utah and a Nevada collection were 2.8 ± 0.8 and 2.2 ± 0.3 mg/kg, respectively. The i.v. LD(50) of methyllycaconitine (MLA), a major toxic alkaloid in low larkspur, was 4.6 ± 0.5 mg/kg, whereas the i.v. LD(50) of a 1:1 mixture of MLA and zygacine was 2.9 ± 0.7 mg/kg. The clinical signs in mice treated with this mixture were very similar to those of mice treated with zygacine alone, including the time of onset and death. These results suggest that there is an additive effect of coadministering these 2 alkaloids i.v. in mice. The results from this study increase knowledge and understanding regarding the acute toxicity of death camas. As combined intoxications are most likely common, this information will be useful in further developing management recommendations for ranchers and in designing additional experiments to study the toxicity of death camas to livestock.

Investigation of the susceptibility of various strains of mice to methyllycaconitine toxicosis.

Although the mechanism of action for larkspur alkaloids has been described, little information is available on the variation of the physiological response of individual animals to larkspur alkaloids. Anecdotal observations and pilot studies in cattle indicate that there is animal-to-animal variation in response to a debilitating dose of larkspur alkaloids. The objective of this study was to determine whether there is variation in susceptibility of different strains of mice to larkspur alkaloid toxicosis and to identify factors responsible for the variation that could then be used as a model for studies in cattle. The acute toxicity of methyllycaconitine (MLA) in 9 different inbred strains of mice was compared. The rank order, from most to least susceptible, was A/J>B10>FVB>BALB/c>C57Bl/6>NZW>C3H>DBA>129. The calculated LD(50) ranged from 3.3+/-0.2 to 5.8+/-0.8 mg/kg of BW. The toxicokinetic profiles of MLA in the susceptible A/J strain and the more resistant 129 strain were compared to determine whether their differences in susceptibility were due to differences in their ability to eliminate MLA. The differences in toxicokinetic variables observed did not explain the differences in susceptibility. The protein expression of various nicotinic acetylcholine receptor (nAChR) subunits was also compared between the more resistant 129 strain and the susceptible A/J strain. The 129 strain of mice had twice the amount of alpha7 nAChR subunit expression as the A/J strain, which was in direct proportion to the approximately 2-fold difference in LD(50). There was also a significant difference (P<0.05) in expression of the alpha3 and alpha5 nAChR subunits between the 129 and A/J strains, with the 129 strain having a greater expression in each case. These data suggest that the increased susceptibility of the A/J mice could be due to a reduced expression of nAChR subunits. Similar analyses need to be made in cattle to determine whether there is a difference between breeds in susceptibility to larkspur poisoning and to identify the factors that regulate their susceptibility to larkspur poisoning. This information would be useful for livestock producers in their breeding, culling, and grazing management programs to reduce or prevent larkspur poisoning on rangelands.

Scrapie resistance in ARQ sheep.

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.

The effect of 7,8-methylenedioxylycoctonine-type diterpenoid alkaloids on the toxicity of methyllycaconitine in mice.

Larkspur plants contain numerous norditerpenoid alkaloids, which include the 7,8-methylenedioxylycoctonine (MDL)-type alkaloids and the N-(methylsuccinimido)anthranoyllycoctonine (MSAL)-type alkaloids. The MSAL-type alkaloids are generally much more toxic (typically >20 times). Toxicity of many tall larkspurs, such as Delphinium barbeyi, has been attributed to its large concentration of MSAL-type alkaloids, including methyllycaconitine (MLA). However, the norditerpenoid alkaloids found in the greatest concentrations in most D. barbeyi populations are either deltaline or 14-O-acetyldictyocarpine (14-OAD), both less toxic MDL-type alkaloids. Although the individual toxicities of MLA, 14-OAD, and deltaline have been determined, the impact (additive or antagonistic) that large concentrations of deltaline or 14-OAD in the plant have on the toxicity of MLA is unknown. Consequently, the effect of MDL-type alkaloids on the toxicity of MLA was compared by using median lethal dose (LD(50)) and toxicokinetic profiles of the brainand muscle from mice receiving i.v. administration of these alkaloids, individually or in combination, at ratios of 1:1, 1:5, and 1:25 MLA to MDL-type alkaloids. The LD(50) for MLA alone was 4.4 +/- 0.7 mg/kg of BW, whereas the coadministration of MLA and deltaline at 1:1, 1:5, and 1:25 resulted in an LD(50) of 2.7, 2.5, and 1.9 mg/kg of BW, respectively. Similarly, the coadministration of MLA and 14-OAD at 1:1, 1:5, and 1:25 resulted in an LD(50) of 3.1, 2.2, and 1.5 mg/kg of BW, respectively. Coadministration of mixtures did not result in increased MLA bioavailability or alterations in clearance from the brain and muscle. Consequently, the increased toxicity of the mixtures was not a result of increased MLA bioavailability (based on the maximum concentrations observed) or alterations in MLA clearance from the brain and muscle, because these were unchanged. These results demonstrate that MDL-type alkaloids have an additive effect on MLA toxicity in mice and may also play a role in the overall toxicity of tall larkspur plants in cattle.

Duodenal somatostatinoma presenting with complete somatostatinoma syndrome.

Somatostatinomas are the rarest pancreatic endocrine tumors and can arise in the pancreas or duodenum. Duodenal somatostatinomas are less common than, and are distinguished from, their pancreatic counterparts by a frequent association with type I neurofibromatosis, the presence of psammoma bodies, the less frequent presence of metastatic disease, and the absence of somatostatinoma syndrome (diabetes mellitus, steatorrhea, and cholelithiasis). We report a case of somatostatinoma with metastases and psammoma bodies presenting with all three features of the syndrome in a patient with neurofibromatosis. Although several reports have documented portions of the syndrome in patients with duodenal somatostatinomas, to our knowledge, this is the first report of the complete syndrome associated with a duodenal lesion.