Effects of Manual Lymphatic Drainage Combined with Press-Release Method Orthopedic Manual Lymphatic Physiotherapy on Upper Extremity Edema and Shoulder Joint Range of Motion and Pain in Patients with Breast Cancer- Juniper Publishers

 

Journal of Surgery- Juniper Publishers

Abstract

Purpose: The purpose of this study is to investigate the effectiveness of orthopedic manual lymphatic drainage techniques to move fluid and soften hardened tissues using functional assessment of the upper extremity of patients after breast cancer surgery, as well as edema and pain scales.

Methods: The study included 24 patients diagnosed with lymphedema following mastectomy surgery, who received the intervention twice a day, three times a week for six weeks, and were evaluated for upper extremity swelling volume assessment and shoulder joint range of motion and pain sensory.

Results: In conclusion, this study demonstrates that the integrated lymphatic therapy approach of orthopedic manual lymphatic physiotherapy is an effective treatment for reducing edema, improving shoulder joint range of motion, and reducing pain sensory in the upper extremity in postoperative patients with breast cancer.

Conclusion: Orthopedic manual lymphatic physiotherapy with press-release techniques was effective in improving upper extremity edema, shoulder joint range of motion, and pain in a post-breast cancer surgery patient.

Keywords: Edema; Manual Lymph Drainage; Orthopedic manual lymphatic physiotherapy; Interstitial fluid; Alpha waves

Abbreviations: CDT: Complete Decongestive Therapy; DASH: Disabilities of the Arm, Shoulder, and Hand; NRS: Numeric Rating Scale; MLD: Manual Lymphatic Drainage; MWF: Mobilization with Facilitation; KPIMT: Korea Pediatric Integrative Manual Therapy; SF-MPQ: Short-Form McGill Pain Questionnaire

Introduction

In recent years, breast cancer is ranked second in the five-year cancer prevalence rate among women in Korea with 21.9% per 100,000 population, and the incidence of breast cancer has also increased rapidly, ranking first among newly diagnosed malignant neoplasms in women with 20.3%, and by age group, it is ranked first in the 35-64 age group with 29.1%, and the five-year survival rate is 96.8%, showing a high survival rate [1].

Lymphedema, which occurs after cancer progression and treatment, was found to occur in more than 50% of breast cancer patients [2], who underwent surgery to remove the lymph nodes in the armpit and radiation therapy, and in more than 64% of patients who underwent surgery to remove the lymph nodes in the groin or pelvis [3]. Lymphedema is a soft tissue swelling caused by the accumulation of proteinaceous fluid in the pericellular space of cells. This reduces the carrying capacity of the lymph and increases the lymphatic load. The severity of lymphedema is graded using the International Society of Lymphology’s scale [4]. Lymphedema is a chronic, progressive condition in which the swollen area increases in size and weight, limiting motion and joint movement with postural changes and pain in daily activities. Conservative treatment with radiation therapy is associated with decreased shoulder joint range of motion, localized dysfunction, and the development of lymphedema [5].

In addition, lymphedema occurs in about 20 to 40 percent of patients after surgery, such as mastectomy and lymph node dissection, and is associated with upper arm dysfunction, including numbness, pain, limited range of motion in the shoulder joint and neck, tendonitis of the rotator cuff, and decreased muscle strength [6,7]. In recent years, some researchers have been working on controlled studies to determine the effectiveness of orthopedic manual physical therapy with Manyal Lymph Drainage (MLD), but the results are still controversial. Multiple Continuous compression is accepted as an essential part of treatment [8]. This effect has been shown to reduce edema by 7-17 % when compression methods such as compression stockings are applied without any other treatment [9].

MLD improves the appearance of edema by improving microvascular tone and increasing mobility, but because MLD is usually performed in conjunction with compression, it is difficult to conclude that it is effective in reducing edema by more than 60%. It would be unreasonable to attribute this effect to MLD alone, The Other Study demonstrated a 20% reduction in edema with a low-elastic bandage and orthopedic manual physical therapy with MLD, while Johansson et al. validated the effectiveness of orthopedic manual physical therapy with MLD alone with a statistically significant difference of more than 7% in favor of MLD over bandage alone [10,11].

Therefore, this study aimed to investigate the effects of applying orthopedic manual physical therapy with MDL technique using upper extremity orthopedic manual physical therapy using mobilization with Facilitation technique and Press - Release technique, a new physiotherapy treatment method, on changes in upper extremity swelling circumference, shoulder joint range of motion assessment, and pain sensation in breast cancer patients, and to provide a basis for future research on physiotherapy intervention programs.

Methods

Participation

In this study, 24 patients who were diagnosed with lymphedema after mastectomy surgery at J Hospital Cancer Specialized Rehabilitation Center located in Jeollanam-do were included in the study from the beginning of June 2023 to the end of March 2024, and the circumference of the swelling area and the contralateral upper limb differed by more than 2 cm, the skin condition of the upper limb was not problematic for applying the bandage method, and the patient was able to perform upper limb exercises according to the therapist’s instructions.

Inclusion criteria for the study were:

 Diagnosed with unilateral lymphedema.

 No active cancer.

 No joint movement restrictions.

 Not taking any medications, foods, etc. that may affect the reduction of edema, such as diuretics that may affect edema reduction.

 No neurological conditions that could affect measurements.

 Guardian has agreed to participate in the study.

Before participating in this study, all subjects were fully informed of the purpose, content, and methods of the study, and only those who voluntarily signed an informed consent form were included in the study. Patients with any of the following conditions were excluded from the study.

 Individuals with cardiac dysfunction and acute thrombosis.

 Individuals with dermatitis.

 Those with acute malignant lymphedema.

 Subjects with arm paralysis and vascular disorders.

 Participation in a study like this study within the last 1 year.

Measures and Procedure

This case study was conducted after obtaining informed consent in accordance with the Declaration of Helsinki. The procedure of this study is as follows (Figure 1). The G-Power 3.0 program (IBM Inc., USA) was used to determine the sample size of this study. To calculate the sample size, the significance level (α) was selected as 0.05, the power (1-β=0.8) was selected as 0.48, and the effect size (d) was calculated using the preliminary experiment.

A minimum of 12 subjects were required for each group, for a total of 24 subjects. The subjects were randomly assigned to the MLD group and the control group. The subjects were randomly assigned and grouped using randomization software (Random allocation software version 1.0, University of Medical Sciences, USA). In this study, the intervention was delivered three times a week, twice a day for six weeks, for a total of 36 sessions. The pretest included general characteristics, upper extremity edema assessment, shoulder joint range of motion, and pain sensation (Figure 2).

Assessment of Edema of the Upper Extremities

When Assessment upper extremity (Figure 2) edema, upper extremity circumference was measured 10 cm below the patient’s elbow joint with a measuring tape (Arm Circumference Gauge, Sammons Preston, USA), a method proven reliable in Taylor’s experiment [12].

Assessment of Range of Motion of Shoulder Joint

To observe the range of motion of the upper extremity before and after treatment, the shoulder joint range of motion was tested using a goniometer. Active flexion, abduction, and external rotation of the shoulder joint were measured in the edema position. The flexion of the shoulder joint was measured while lying supine, bending the knees and hip joints, and placing the feet flat on the floor to prevent hyperextension of the lumbar spine. The elbow joint was extended, and the forearm and palm were kept supinated. The axis of the joint goniometer was aligned with the acromion process of the scapula, which passes through the humeral head. The fixed arm was positioned at the mid-axillary line of the torso, and the movable arm was measured after being positioned at the outer midline of the humerus.

Shoulder joint abduction was measured in the supine position with knees and hips bent and feet flat on the floor. The arm being measured was placed in an anatomical position, and the elbow joint was maintained in an extended state. The axis of the joint goniometer was aligned with the anterior part of the acromion process of the scapula through the center of the humeral head. The fixed arm was parallel to the midline of the sternum and placed on the lateral surface of the anterior surface of the ribcage, and the movable arm was parallel to the midline of the humerus and placed on the anterior surface of the arm before measurement. External rotation of the shoulder joint was measured in the supine position with the knees and hip joints flexion and the feet flat on the floor.

The axis of the joint goniometer was aligned so that the direction of the humeral head coincided with the olecranon process of the ulna passing through the humeral body. The fixed arm was positioned perpendicular to the floor, and the movable arm was measured after being positioned on the ulnar body in the direction of the styloid process of the ulna [13].

Assessment of Pain sensory

The short-form McGill pain questionnaire (SF-MPQ) was used to measure pain sensory, and the reliability of the measurement tool was r=.89.14 The SF-MPQ consists of a total of 15 questions, including 11 questions in the sensory domain and 4 questions in the emotional domain, and can evaluate the sensory and emotional components of pain [14,15]. Each item is rated on a 4-point Likerd scale from 0 (no pain), 1 (mild pain), 2 (moderate pain), and 3 (severe pain). The higher the summed score, the higher the pain sensation. A physical therapist explained to the subjects how to fill out the questionnaire, and self-assessed the questionnaire while the subjects were stable.

Intervention

Experimental Group

The intervention for the MLD technique of the Press-Release technique was administered twice a day, three times a week for six weeks, and was applied for 20 minutes each. The subject was asked to lie down on the treatment table, place a pillow under the knees, bend the hip joint at 70°, and relax the muscles as much as possible. Korea Pediatric Integrative Manual Therapy (KPIMT) Press Release Therapy and Mobilization with Facilitation therapy [16,17]. KPIMT muscular skeletal factor kalten born segmental movement and mulligan concept where there is an arbitrary restriction of movement of the incorrect musculoskeletal system [18,19].

The coordinative control of the use of the arms and hands mixed with the spine, through the handling input of the therapist, with the distribution of various contacts, sufficient proprioceptive and somatosensory input, and through the guiding and assisting of the hand, the sensor light is sufficiently turned on. Mobilization with Facilitation (MWF) technique and stretching release technique and press release technique are applied to each joint junction for inputting somatosensory and proprioceptive sensory information of the muscle when providing sufficient support for the ability to recover balance from touch to come.16 Provides joint stability and Recovers pain by stimulating the parasympathetic nerve and increasing lymph vessel flow [16].

PR technique treatment area and pressure stage; The pressure level of the treatment is divided into skin surface, dermis layer, fascia surface, and submuscular area according to the depth, and the level of force is divided into 1-4 levels, changing depending on the treatment area.

Before all lymphatic physical therapy, pump Jugular angle area 25 times before performing the next treatment [16].

This section deals with the axillary group and hand arm region. the muscle each group muscles (apex, base, medial lateral posterior, anterior). ① Effleuage→ ② Treatment of the Apex part of infraclavicle lymph nodes → delto-pecto lymph nodes → axillary lymph nodes and trapeziurs and deltoid muscle (pressrelease technique 20 ) → ③ Treatment of the Base part of Latissimus dorsi and teres major (press-release technique 15) → ④ Treatment of the Medial Border part of serratus anterior and inter costalis muscle (press-release technique 15) → ⑤ Treatment of the lateral border part of Coracobrachialis and bicepce muscle (press-release technique 15) → ⑥ Treatment of the Anterior wall of Pecto major and minor and subclavicles muscle with anterior wall Cord bending technique (press-release technique 15) → ⑦ Treatment of the posterior wall of Latissimus dorsi and teres minor muscle (press-release technique 15) → ⑧ External Rotation Techniques to increase scapulo-humeral joint range of motion → ⑨ Posterior wall Cord bending technique → Pump stroke stage of Upper arm (Deltoid → Biceps → Triceps 15section) and Lower arm (Supnation and Pronation Scoope stroke) → Hand Pump ans thumb circle strock→ Ocillization ⑩ Effleuage (Figure 3).

In the afternoon section treatment, the upper limb bandaging method was performed for 20 minutes followed by a 10-minute rest. The bandaging method applied to the patient’s upper limb was to fold a 4 cm bandage in half, wrap it loosely around the finger, and then apply a cotton stockinette to the upper limb. I wound it on. Afterwards, an undercast pad was added to the wrapped from the fingers upward to the upper limb [20]. For the compression bandage, Lohmman and Rauscher’s low elastic bandage (Germany) was used in both the experimental and control groups.

Control Group

The control group intervention was performed three times a day, every other day for six weeks, with subjects in the supine position on the treatment table, as relaxed as possible, receiving 20 minutes of multi-chamber pneumatic compression per intervention, followed by 10 minutes of relaxation for 10 minutes. The pressure was kept below 30mmHg to prevent further damage to the lymphatic system in the upper extremity. In the afternoon section, the treatment consisted of 20 minutes of upper extremity bandaging followed by 10 minutes of rest. In the afternoon section, bandaging was performed in the same way as in the experimental group.

Statistical Analysis

The data collected in this study were subjected to statistical analysis using SPSS for windows (version 21.0). Descriptive statistical methods were used to determine the subject’s age, weight, height, medical treatment, and area of edema, and an independent samples t-test was performed to determine the homogeneity of the experimental and control groups. For the differences in arm circumference, shoulder joint range of motion, and pain sensation for comparison of edema volume before and after treatment between the experimental and control groups, a Wilcoxon rank test was performed to test the significance before and after treatment of each group, and the differences between each group were performed. To find out, the Mann-Whitney U test was performed. The significance level was set at 0.05.

Results

The total number of subjects in the study was 24 women, and the medical characteristics of the subjects in the study were as follows: 12 (45%) of the 24 subjects received combined radiotherapy and chemotherapy, 9 (40%) received chemotherapy, and 3 (15%) received radiotherapy. An independent samples t-test was performed to test the homogeneity of the experimental and control groups. The location of the edema in the study included 16 (75%) left upper extremity, 8 (35%) right upper extremity, and 24 (100%) had edema in both proximal and distal parts of the upper extremity (Table 1).

Pre-post Treatment Upper Extremity Volume Changes to Assess Edema in Experimental and Control Groups: The volume of the upper arm and forearm of the experimental group decreased significantly from pre-treatment to post-treatment. In the control group, the decrease was significant for the upper arm but not for the forearm (Table 2) (p<0.05).

Compare the Range of Motion of the Shoulder Joint: Changes in pre-post treatment range of motion of the shoulder joint between the experimental and control groups There were statistically significant differences in the pre-post treatment range of motion of the shoulder joint between the experimental and control groups for flexion, abduction, and external rotation, and only for abduction in the control group (p<0.05) (Table 3).

Comparison of Pain Sensory: The changes in pain sensation of the experimental group and control group according to the intervention are as follows (Table 4). The pain sensation before and after the intervention for each group was compared by paired sample t-test, and there was a statistically significant difference after the intervention in the experimental group (p<.05) (Table 4).

Discussion

This study aims to explore a manual lymphatic drainage (MLD) therapy program that incorporates upper extremity orthopedic lymphatic physiotherapy, which is more effective than compression methods used in physical therapies for the therapeutic management of upper extremity lymphedema following mastectomy.

Specifically, this study evaluates effects on edema, range of motion, and pain sensation in the upper extremities of patients with lymph edema. The overall mean age of the participants was 52.25±8.96 years; this is slightly higher than the findings of the Korea Central Cancer Registry, reporting the highest incidence of breast cancer in women aged 40-49 years in Korea.

However, this could be attributed to the random selection of patients within a specific period and the overall incidence rate is expected to align with the Registry’s findings [21]. Tumor locations were 55.0% left-sided and 45.0% right-sided, aligning closely with the findings reported by the Korean Breast Cancer Society, with 51.6% left-sided, 47.5% right-sided, and 0.9% bilateral [22]. Lee and Bae [23] also reported a similar pattern, with a higher number of patients with left-sided breast cancer than those with right-sided [23]. According to the American Cancer Society, one in eight American women is expected to develop breast cancer, with incidence rates being exceptionally high among women aged 50-59 years, like the findings of the Korea Central Cancer Registry [24].

In addition, Siotos [25] found the upper outer quadrant of the breast at a 36.2% incidence as the most common tumor location, followed by the upper inner quadrant at 13.1%, the lower outer quadrant at 9.8%, the lower inner quadrant at 7.2%, the nipple at 1.2%, the axillary tail at 0.3%, and overlapping regions at 24.7% [25]. Post-surgical treatment among the 24 participants included radiation and chemotherapy in 45%, chemotherapy alone in 40%, and radiotherapy alone in 15%, with radiation identified as a significant risk factor for lymphedema [26]. However, this study selected patients exhibiting lymphedema on both the proximal and distal sides of the upper extremity that differed by more than 2 cm from the normal side, irrespective of their medical treatment type, limiting the correlation between treatment method and lymphedema occurrence in the participants.

The results of this study showed a significant decrease in pain sensation after intervention (p<.05). The lymphatic system plays a crucial role in maintaining homeostasis of macromolecules, lipid absorption, immune function, and interstitial fluid regulation. Its main feature is the ability to remove interstitial fluid and proteins from the interstitial space, draining them from the interstitial space into the vasculature [27]. In their study on the use of physiotherapy and exercise therapy after mastectomy in women aged 65 years and older, Tunay et al [28], found significant reductions in pain and improvements in shoulder function, range of motion during flexion, abduction, and external rotation, muscle strength, physical function, and quality of life, as well as a decrease in lymphedema volume [28]. In another study, Cho et al [29], compared conventional physical therapy with manual lymphatic drainage applied three times a week for four weeks in 41 patients with lymphedema and a pain scale score of 3 or higher [29]. They found significant improvements in functional and symptomatic aspects, shoulder flexor muscle strength assessed by the DASH (disabilities of the arm, shoulder, and hand) score, and pain levels measured by the NRS (numeric rating scale). Moreover, they noted a more significant difference in the group receiving combined conventional physical therapy and manual lymph drainage compared to those receiving conventional physical therapy alone, which is consistent with the results of this study.

MLD techniques enhance the movement of lymphatic fluid in the interstitial space, contributing to the removal of interstitial fluid. This drainage into the venous system improves venous return, reduces systemic vascular resistance, and facilitates oxygen delivery to cells. In other words, the improved blood circulation after the intervention is believed to facilitate the muscle cell oxygenation, consequently alleviating pressure on the muscles and, thus, contributing to the reduction in muscle tone [30]. Continuous light stimulation during moderate pressure massage activates Aβ fibers, stimulating the substantia gelatinosa in the spinal dorsal horn and inhibiting pain transmission [31]. Additionally, oxytocin’s activation of the endogenous pain control system induces analgesic effects [32,33]. This study suggests that the press-release rhythmic stimulation effectively reduced pain in patients with breast cancer.

Research suggested post-surgical exercises for patients with breast cancer to reduce complications and improve physical function, including flexibility exercises to smooth joint range of motion, muscle strength exercises, and exercises to strengthen cardiorespiratory function and increase stamina. [34] A similar study compared the effectiveness of MLD and complete decongestive therapy (CDT); this physical therapy regimen includes compression bandaging in moving fluid and reducing limb volume in 13 patients with lymphedema of the lower extremities. The study found that the volume of the leg and the amount of intracellular and extracellular fluid decreased when using the physical therapy regimen compared to CDT [35]. Orthopedic manual lymphatic physiotherapy integrated with MLD influences the body through various mechanisms. It has been reported to reduce excessive sympathetic nervous system activity due to stress or other factors and enhance parasympathetic nervous activity, promoting relaxation. Additionally, it increases the activity of alpha waves, associated with relaxation and calmness, and decreases gamma waves, activated during extreme arousal and excitement [36]. This study suggests that the press-release rhythmic stimulation used in orthopedic manual lymphatic physiotherapy, akin to functional massage, facilitates muscle relaxation and joint mobility as well as optimally applies lymphatic treatment. This approach improved the range of motion and reduced pain in the shoulder joint, as evidenced by the study’s results.

However, this study has certain limitations stemming from the number of local participants and the control of external variables. Future research should involve a larger sample size and more controlled measurement of various variables.

Conclusion

This study investigated the effectiveness of orthopedic manual lymphatic physical therapy using the press-release technique in improving upper extremity swelling, shoulder joint range of motion, and pain in patients after breast cancer surgery.

 The volume of the upper arm and forearm of the experimental group decreased significantly from pre-treatment to post-treatment. In the control group, the decrease was significant for the upper arm but not for the forearm (p<0.05).

 Changes in pre-post treatment range of motion of the shoulder joint between the experimental and control groups There were statistically significant differences in the pre-post treatment range of motion of the shoulder joint between the experimental and control groups for flexion, abduction, and external rotation, and only for abduction in the control group (p<0.05).

 The changes in pain sensation of the experimental group and control group according to the intervention are as follows (Table 4). The pain sensation before and after the intervention for each group was compared by paired sample t-test, and there was a statistically significant difference after the intervention in the experimental group (p<.05).

Orthopedic manual lymphatic physiotherapy with pressrelease techniques in manual lymph drainage was effective in improving upper extremity edema, shoulder joint range of motion, and pain in a post-breast cancer surgery patient.

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Retatrutide as a Novel Treatment for Obesity and Type 2 Diabetes- Juniper Publishers

 

Diabetes & Obesity- Juniper Publishers

Abstract

Retatrutide is a triple hormone agonist of the receptors of glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide (GIP) and glucagon that can be administered subcutaneously once a week. Different doses and escalation schedules of retatrutide were evaluated in 2 phase 2 American trials of subjects with obesity (n=338) and type 2 diabetes (n=281). In the obesity trial of 48 week-duration, percentage change in body weight from baseline after 24 weeks and 48 weeks were the primary and secondary outcomes respectively. At 24 weeks, the mean percentage decrease in weight ranged from 7.2% to 17.5% across retatrutide groups versus 1.6% with placebo. At 48 weeks, the decrease was 8.7% to 24.2% versus 2.1% with placebo. In the diabetes trial, the primary endpoint was a change in glycated hemoglobin (HbA1c) values from baseline to week 24. This trial included an active treatment group receiving dulaglutide 1.5mg once weekly. Secondary endpoints included change in HbA1c and body weight at 36 weeks. At 24 weeks, mean HbA1c reductions were 0.43% to 2.02% with retatrutide, 1.4% with dulaglutide and 0.01% with placebo. At 36 weeks, mean percentage weight reductions were 3.2% to 16.9% with retatrutide, 2.0% with dulaglutide and 3.0% with placebo. While the decrease in HbA1c levels reached a plateau at 24 weeks, weight loss in the 2 studies was progressive without evidence of attenuation effect up to the end of follow-up at 36-48 weeks. The most common adverse effects of retatrutide were gastrointestinal (GI) such as nausea, diarrhea, vomiting and constipation reported by 13-50% in the retatrutide groups compared with 35% in the dulaglutide group, and 13% in the placebo group. Discontinuation rates due to adverse effects were also higher with retatrutide being 16-17% compared with 2% with dulaglutide and 0-4% with placebo. In summary, retatrutide is highly effective drug in causing substantial weight loss and HbA1c reduction. Yet, tolerance to this drug seems sub-optimal. Phase 3 trials are urgently needed to clarify efficacy and safety of retatrutide.

Keywords: Retatrutide; GLP-1; GIP; Glucagon; Obesity; Diabetes and tirzepatide

Introduction

Retatrutide (LY3437943) is a novel triple agonist of the 3 receptors GLP-1, GIP, and glucagon [1]. Compared to the native hormones, retatrutide shows 2.9-fold less potency at the glucagon receptor (GCCR), 2.5-fold less potency at the GLP-1 receptor, and 8.9-fold greater potency at human GIP receptor [1]. The approximate half-life of retatrutide is approximately 6 days making it suitable for once weekly administration [2]. Pre-clinical studies and phase 1 trials showed that retatrutide was highly effective in promoting weight loss and lowering HbA1c levels [1,2]. Recently, 2 phase 2 studies designed to examine efficacy and safety of retatrutide for treatment of obesity and type 2 diabetes were published (Table 1) [3,4]. The main purpose of this article is to provide an appraisal of this novel agent based on available data.

Retatrutide for Treatment of Obesity

Retatrutide, in different subcutaneous doses of 1, 4, 8 and 12mg and escalation schedules, was evaluated for treatment of obesity in a phase 2, randomized, placebo-controlled, double-blind trial of 48 week-duration in the USA (Table 1) [3]. Contrary to most preceding obesity trials that predominantly enrolled women, this study included 51.8% men [3]. Participants (n=338) had mean age of 48.2 years, and mean baseline weight of 107.7kg, and body mass index (BMI) of 37.3kg/m2 (Table 1) [3]. All subjects received lifestyle intervention including regular counseling sessions [3]. At 24 weeks, mean percentage decrease in weight (primary outcome) ranged from 7.2% to 17.5% with different retatrutide doses compared with 1.6% with placebo. At 48 weeks, there was progressive decrease in weight (secondary outcome) ranging from 8.7% to 24.2% with retatrutide versus 2.1% with placebo [3]. Weight loss was dose-related between retatrutide doses of 1 to 8mg. However, the difference in weight loss between the highest 2 doses, 8 and 12mg, was minimal [3]. Inspection of the trajectory of weight loss with time showed that weight loss with retatrutide was evident after 4-8 weeks and progressed with no evidence of plateau to the end of follow-up at 48 weeks [3]. At 48 weeks, weight reduction of ≥15% was achieved by 75-83% of subjects who received 8-12mg dose of retatrutide. Interestingly, women and subjects with BMI of ≥35 kg/m2 had more weight loss than men and subjects with BMI < 35kg/m2 [3]. Thus, among women randomized to the highest retatrutide dose, percentage mean weight loss at 48 weeks was 28.5% versus 21.9% among men. In subjects with baseline BMI ≥35 kg/m2, corresponding weight loss was 26.4% versus 21.5% in those with BMI < 35kg/ m2 [3].

Retatrutide for Treatment of Type 2 Diabetes

In another phase 2 trial in the USA, retatrutide was evaluated for treatment of type 2 diabetes in comparison with subcutaneous dulaglutide (1.5mg/weekly) and placebo (Table 1) [4]. The study duration was 36 weeks. The primary outcome was change in HbA1c values from baseline to 24 weeks, whereas secondary endpoints were changes in HbA1c and weight after 36 weeks (Table 1) [4]. Patients (n=281, 56% women, mean age 56 years) had type 2 diabetes of approximately 8 year-duration uncontrolled on metformin with mean baseline HbA1c of 8.3% [4]. At 24 weeks, reductions in HbA1c levels were dose-related in the retatrutide groups ranging from 0.43 to 2.02%, versus 1.31% in the dulaglutide group and 0.01% in the placebo group [4]. HbA1c reductions were significantly greater with the 2 retatrutide highest doses (8mg given in slow escalation and 12mg/week) than with dulaglutide (P<0.002) [4]. At 36 weeks, a similar trend was observed but with minimal changes in HbA1c levels beyond 24 weeks in all groups [4].

There was dose-related weight loss among the retatrutide groups at 36 weeks that ranged from 3.2-16.9% compared with weight loss of 3.0% in the placebo group, and 2.0% in the dulaglutide group [4]. Contrary to the decrease in HbA1c values that attained a plateau after 24 weeks, weight reduction with retatrutide continued to progress to the end of follow-up at 36 weeks [4]. Moreover, approximately 60% of patients randomized to high dose retatrutide (8-12mg) lost ≥ 15% of weight compared with 2% with placebo [4]. It should be emphasized that exceeding the cutoff weight loss of 15% is clinically important. Indeed, sustained weight reduction of 15% or more in patients with type 2 diabetes may induce remission in a large proportion of subjects and improve metabolic status in the remaining patients [5]. Meanwhile, the magnitude of weight loss with use of retatrutide was less pronounced in patients with type 2 diabetes at 36 weeks compared with obese subjects without diabetes at 48 weeks in the obesity trial (Table 1) [3]. This observation may be due to inclusion of younger patients with more severe degree of obesity at baseline, and longer follow-up in the obesity trial compared with the diabetes trial (Table 1). In addition, for other unclear reasons, obesity drugs were generally less effective in patients with type 2 diabetes than in in people without diabetes [5].

Effects of Retatrutide on Cardiovascular Risk Factors

Amelioration of several cardiovascular risk factors, mainly due to weight loss, was observed with retatrutide treatment [3,4]. In the obesity study, 72% of the participants who had prediabetes at baseline in the retatrutide groups reverted to normoglycemia (HbA1C < 5.7%) as compared with 22% of individuals in the placebo group [3]. In addition, the decrease in systolic blood pressure/diastolic blood pressure (SBP/DBP) was 8.8/2.8mmHg versus 2.9/1.0 in placebo at 36 weeks [3]. In the diabetes study, SBP decreased by up to 8.8mmHg with retatrutide versus a reduction of 1.5mmHg with dulaglutide, and an increase of 1.5mmHg with placebo [4]. In the obesity study, retatrutide decreased fasting levels of plasma triglycerides by up to 40%, and low-densitylipoprotein cholesterol (LDL-C) by up to 22% at 48 weeks [3]. In the diabetes trial, retatrutide decreased plasma triglyceride by up to 35% at 36 weeks compared with 4% reduction with dulaglutide and 9% with placebo [4]. Changes in circulating levels of LDL-C, high-density lipoprotein cholesterol and free fatty acids were not statistically different between various patient groups in the diabetes study [4].

Safety of Retatrutide

In the 2 phase 2 trials, 16-17% discontinued retatrutide compared with 0-4% with placebo due to adverse effects (Table 1) [3,4]. In general, safety profile of retatrutide mimics that of incretin-based drugs with GI adverse effects being the most common in incidence and the most frequent cause of drug discontinuation. GI adverse effects (nausea, diarrhea, vomiting and constipation) were dose-related and occurred mainly during the early dose escalation period [3,4]. In the obesity trial, proportions of subjects reporting nausea were 14-60% with retatrutide versus 11% with placebo [3]. In the diabetes study, GI adverse effects were reported by 13-50% in the retatrutide group, 35% in the dulaglutide group, and 13% in the placebo group [4]. This relatively high rate of GI adverse effect may be attributed to the glucagon receptor agonism component of retatrutide. In fact, glucagon has been shown to slow gastric emptying and inhibit GI motility [1].

Effect of Retatrutide on Heart Rate

Increase in heart rate, of approximately 2-6 beats per minute (bpm) compared with placebo, was observed with use of all GLP-1 receptor agonists and with the dual GLP-1/GIP receptor agonist tirzepatide [6-11]. However, this adverse effect did not result in an increase in cardiovascular events in dedicated randomized trials [7,8]. With retatrutide, the increase in heart rate peaked 24 weeks after starting treatment and partially declined thereafter [3,4]. Placebo-adjusted increase in heart rate by retatrutide was 5.6bpm in the obesity trial at 48 weeks and 7.5bpm in the diabetes trial after 36 weeks [3]. In the latter study, the corresponding increase was 5bpm with dulaglutide. Furthermore, cardiac arrhythmias occurred in 4-14% and 2-3% in the retatrutide groups and placebo group, respectively in the 2 trials [3,4]. The increase in heart rate by teratrutide could be mediated in part by glucagon receptor agonism. Indeed, most, but not all, studies have shown that glucagon administration increases heart rate acutely in humans [12].

Advantages of Retatrutide

The major advantage of teratrutide is its high efficacy for weight loss that exceeds efficacy of all available anti-obesity drugs. The dual GLP-1 and GIP agonist tirzepatide is currently considered the most effective drug to lower HbA1c levels and promote weight loss [10,11]. While no direct comparison between retatrutide and tirzepatide is available and duration of use is different (36- 48 weeks with retatrutide versus 72 weeks with tirzepatide), retatrutide may be superior to tirzepatide in both parameters, particularly weight loss (Table 2) [3,4,10,11]. Furthermore, the difference in efficacy of weight reduction between the 2 agents may become even greater with more prolonged use of teratrutide beyond 48 weeks. This enhanced efficacy of retatrutide in promoting weight loss is most likely mediated through glucagon receptor agonism. In fact, Goscun et al. [1] have shown that retatrutide caused weight loss in obese mice by increasing energy expenditure through glucagon receptor engagement. Moreover, a phase 1 study has shown that retatrutide may decrease appetite and increase sensation of fullness and satiety in patients with type 2 diabetes [2]. Decreased appetite was reported by 11-31% among retatrutide-treated subjects (vs 9% placebo) in the obesity trial and by 10% (vs 2% placebo) in the diabetes trial [3,4].

Limitations of Retatrutide

The main limitation of retatrutide is the high frequency rates of discontinuation due to adverse effects reaching 17% with the highest doses. These rates are much higher than the 7% discontinuation rate reported with the highest dose of tirzepatide (15mg/week) versus 4% with placebo [10]. Since most GI adverse effects occurred during retatrutide dose escalation, the use of smaller starting doses and slower titration might decrease the incidence of GI adverse effects. In addition, the increase in pulse rate and arrhythmias is concerning and should be inspected thoroughly in pending trials, preferably using Holter monitoring of heart rate.

Conclusions and Current Needs

No doubt, retatrutide is a promising novel tri-agonist of receptors of the 2 incretins GLP-1 and GIP and glucagon. This drug showed high efficacy in causing weight loss not previously recorded with any anti-obesity agent. Additionally, its efficacy for treatment of type 2 diabetes is at least comparable to tirzepatide (Table 2). Meanwhile, tolerance to retratrutide may be suboptimal with unacceptable high discontinuation rates due to adverse effects. Moreover, there are signals of increased incidence of cardiac arrhythmias associated with its use. Ongoing large-scale phase 3 trials should clarify efficacy and safety of retratrutide in subjects with obesity and type 2 diabetes. In addition, adequately powered and long-term (at least 3 years) studies designed to see the effects of retatrutide on cardiovascular events and mortality should be conducted.

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Residual effects of Lithium in Muscle and Organ Tissues of Sheep Post-Ingestion of Lithium Chloride- Juniper Publishers

 

Dairy & Veterinary Sciences- Juniper Publishers

Abstract

Conditioned taste aversions (CTA) occur when animals associate gastrointestinal distress with a particular food source. CTA strategy can be used to reduce animal consumption of an undesirable feedstuff. Lithium chloride (LiCl) has been used in wild ungulates as a CTA and could be used in white tail deer (WTD) as a potential CTA. Consumption of LiCl by WTD may leave residue in meat which may be consumed by a human. The objectives of this study were to examine the effect of dietary LiCl on kinetics and depletion of lithium in muscle, kidney, and liver tissue in adult domestic sheep (model for WTD). In experiment1, eleven adult sheep orally received either 150 mg LiCl/kg BW (n = 8) or placebo (n = 3). Using aseptic procedure, muscle biopsies were taken at 4,8,12,24,48,96,192, and 240 hours post LiCl ingestion and lithium concentrations were measured. In experiment2, sixteen adult sheep received either 450mg LiCl/kg BW (n = 14) or placebo (n = 2). In experiment3, nine adult sheep orally received a single dose of 150mg/kg BW. Three-animal groups were euthanized at 7,24, and 96 hours post-LiCl ingestion and muscle, liver, and kidney samples were harvested to measure lithium concentrations. Low dose of LiCl reached a maximum level in muscle 24 hours post-ingestion and returning to basal levels (P = 0.72) by 192 hours. High mortality (12 of 14; 86%) occurred following high dose administration resulting in an inability to determine maximum concentration levels or appreciate differences between muscle, organ tissue types and over time. Lithium concentrations were greater (P<0.01) in liver and kidney compared to muscle at 7 and 24 hours post ingestion, but no difference in lithium concentration was detected in the three tissues at 96 hours (P > 0.05). It appears that a withdrawal period in muscle tissue for low dose LiCl in domestic sheep is 192 hours. The toxic threshold for domestic sheep, and likely other small ruminants, occurs between 150-450mg LiCl/kg body weight.

Keywords:Crop depredation, Deer, Lithium chloride, Lithium chloride toxicity, Small ruminants, Taste aversion

Abbreviations:CTA: Conditioned Taste Aversion; WTD: White-tailed deer; LiCl: Lithium Chloride

Introduction

White-tailed deer (Odocoileus virginianus; WTD, hereafter) are one of the most widespread large mammal species of North America, with correspondingly large impacts on society, both positive (e.g., hunting, wildlife viewing) and negative (e.g., car collisions, crop depredation). White-tailed deer inhabit a variety of areas, occurring almost where digestible forage is available and accessible habitat cover is nearby. In recent years, population numbers have drastically increased in many areas of the Western United States, potentially due to their extreme adaptability and versatility [1]. High population densities have been thought to increase dispersal and movement rates, likely causing them to travel further across the landscape in search of available resources [2]. As deer movement and dispersal rates increase, more encounters with agricultural fields containing nutritious crops occur [2], resulting in an increase in crop depredation rates.

To mitigate costs associated with abundant deer while maintaining recreational and economic benefits, there is a pressing need to find effective deer deterrents. In the past, multiple deterrent methods targeted at reducing deer damage have been tested, including propane exploders and other frightening devices, fencing, and lethal removal [3-5]. Although previously tested deterrents have resulted in a wide range of effectiveness, wildlife managers are still searching for a deterrent method that is cost-effective with high efficacy rates across a multitude of wildlife species. One promising method yet to be tested in an open field setting for deterring WTD is the use of lithium chloride (LiCl), a gastrointestinal toxicant that has successfully been used to create taste aversions to specific food items in both carnivores and ruminants.

Previous studies have shown high efficacy in reducing the amount of food consumed after LiCl was ingested as treated animals associated targeted food sources with gastrointestinal distress [6-9]. However, most of these studies were conducted in controlled, captive feeding trials where ruminants, as well as carnivores, were given the choice to consume food items pre- and post-ingestion of LiCl [8-11]). Due to LiCl creating strong taste aversions across multiple species, it has a potential of being a successful deterrent method in reducing WTD crop depredations.

Before implementation of LiCl as a depredation deterrent in an open field setting can be utilized, key issues regarding toxicity and accumulation in deer tissues needs to be addressed. One challenge with using LiCl is that crop depredation season overlaps with hunting season in many parts of WTD habitat range (i.e., late summer through fall). As a result, it is important to first understand withdrawal factors in different types of animal tissues that may be consumed by humans. Information regarding LiCl and pharmacokinetic data in small ruminants to LiCl is lacking, which compelled the need for this study prior to using LiCl as a deterrent in an open field setting.

Although the eventual intent is to use LiCl as a deterrent on WTD, domestic sheep were used in this study as a surrogate due to logistics and cost. Domestic sheep have been used in a variety of feeding trials to test the efficacy and necessary dosage needed of LiCl to create an effective aversion [12-14]. Higher dosages often result in a greater aversion effect [15], but toxicity levels and tissue withdrawal times have yet to be reported. Thus, we addressed the following research questions:

1) What are the concentration levels of LiCl over time in differing body tissues at a realistic dose range that may be consumed by a deer in an open field setting based on LiCl withdrawal in the sheep model?

2) What is the maximum realistic dosage that could be consumed in a field setting and is this toxic for small ruminants?

Material and Methods

Animal use and protocols were approved by the Institutional Animal Care and Use Committee at the University of Idaho (IACUC-2017-70). The kinetics and toxicity of LiCl was tested using adult domestic sheep located at the University of Idaho Sheep Center in Moscow, Idaho. Suffolk, Targhee, and Targhee/Polypay crossbred sheep were used in this study, and all experiments were conducted at the University of Idaho Sheep Center. All animals were housed in an indoor/outdoor covered barn; feed and water were available ad libitum. Grain was provided once a day after biopsy samples had been collected.

Before each experiment began, sheep were weighed on an electric platform scale (+/- 1 kg), so that the appropriate dosage of LiCl for each experiment and animal could be determined on a per-kg of body weight basis. Subsequently, the appropriate amount of LiCl was dissolved in 240 mLs of cold water, and administered via drenching (i.e., orally inserting a lubricated stomach tube to the level of the abomasum). Control animals were drenched only with 240mLs of cold water minus the LiCl. Three experiments were conducted to analyze and compare lithium concentration in kidney, liver, and muscle tissues at a low (150 mg/kg) and high (450 mg/kg) dosage. In all experiments animals were visually observed for behavioral changes. All tissue samples were analyzed at the University of Idaho toxicology lab.

In the first experiment, eleven adult sheep were used to assess the kinetics and depletion of LiCl in muscle tissue at a 150mg LiCl/kg of body weight dosage, which was considered a low dose [7,9,16]. On the first experimental day each treated sheep (n = 8) was weighed and orally drenched with a single dose of LiCl [7,9]. Controls (n = 3) received a drench of water only. Muscle biopsy samples (~1g per sample) were extracted from the triceps and upper thigh muscle (biceps femoris, vastus lateralis, and semitendinosus) for lithium concentration analysis. Animals were physically restrained during muscle biopsy. Once restrained, the area of biopsy was surgically prepared, and a local anesthetic (Lidocaine 1%) was administered within the area to affect. The skin was incised, and a punch biopsy tool (MiltexS^® 6mm, Princeton, NJ.) was used to remove approximately 1g of muscle sample. Each 1g sample of muscle tissue was placed into a sterile, labeled Whirl- Pak® and frozen until analyses for lithium quantification.

It has been reported that the maximum level of lithium in blood occurs 4-8 hours post-ingestion [17,18], and animals were completely cleared of lithium after 240 hours [18]. Collection of muscle biopsy samples were made at 4, 8, 12, 24, 48, 96, 192, and 240 hours post LiCl ingestion to cover the entire time span between maximum peak levels and complete lithium metabolism.

In the second experiment, sixteen adult sheep were used to assess the kinetics and depletion time at 3x the recommended 150mg LiCl/kg body weight dosage. On the first day of the experiment each sheep was weighed and orally drenched with 450mg LiCl/kg body weight in cold water (n = 14) or just cold water (n = 2). Muscle biopsies were once again collected following the protocol previously described for experiment 1. If an animal died during the trial a necropsy was immediately conducted and 1g of kidney, liver, and muscle samples were each collected from the deceased animal. During the necropsy all other major organs and muscle groups were observed by a veterinarian to determine if the ingested LiCl had resulted in reportable necropsy findings.

In the third experiment, nine adult sheep were used to analyze lithium concentrations within kidney, liver, and muscle tissues, at time intervals surrounding the peak lithium concentration for low dose (150 mg/kg) ingestion. Based on the results from experiment 1, the peak lithium concentration occurred ~25 hours post-ingestion. On day one all sheep were weighed and orally received a single dosage of 150 mg LiCl/kg body weight mixed with 240 mL of cold water. Sheep were terminated using a penetrating cap and bolt system with exsanguination at intervals surrounding peak lithium concentration times. Group 1 (n = 3) were terminated 7 hours post LiCl ingestion, group 2 (n = 3) 24 hours post LiCl ingestion, and group 3 (n = 3) 96 hours post LiCl ingestion. Tissue samples (1g) from the kidney, liver, and muscle were collected from each animal. Field necropsies were conducted to assess any notable findings that may have been related to LiCl ingestion.

To measure lithium concentrations in tissues, a Perkin Elmber^® Optima 8300 Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) was used. The ICP-OES equipment determined the lithium concentration within each tissue sample using plasma and a spectrometer (operating conditions; plasma: 15 L/min, auxiliary: 0.2 L/min, nebulizer: 0.73 L/min, flow rate: 1.5 mL/min, and wash rate: 2.00 mL/min) [19]. Equipment was calibrated with concentrated redistilled trace metal grade nitric acid and water [19]. Tissue samples were frozen until all samples for the trial had been collected and all samples were tested consecutively to avoid recalibrating equipment multiple times. All samples were analyzed on a wet weight basis, and 1g of tissue sample was added to, and mixed with, 3 mL trace metal grade nitric acid in a 10 mL test tube [19]. The tubes were then heated for 6 hours at 30 °C, then 1 hour at 70 °C, and finally for 8 hours at 120 °C [19]. The tubes were then cooled, vortexed, and centrifuged as needed to produce transparent solutions to prevent clogs from occurring within the nebulizer [19]. If particles remained within the solution a 0.45 Acrodisc filter was used to eliminate the remaining particles [19].

Data on the effects of LiCl on tissue lithium concentration were analyzed using a general linear model (GLM) procedure in SAS [20]. The model included the fixed effect tissue types (muscle, live, kidney), time and their two-way interaction with significance declared at P < 0.05. Using SAS GLM, the effect of low dose LiCl on muscle tissue concentration was also analyzed using GLM. The model included the effect of time.

Results and Discussion

Experiment 1:

Feeding low dose (150 mg/kg) of LiCl caused an increase (P < 0.01) in muscle Lithium at hours 4, 8, 12, 24, 48, and 96 as compared with baseline lithium concentrations. At hours 192 and 240 the muscle concentration of lithium was not different from baseline levels at (P ≤ 0.9). Lithium concentration in muscle tissue peaked at (~24 hours post-ingestion (7.8 μg/g, Figure 1). Lithium concentrations declined thereafter and reached baseline level at 192 hours post-ingestion.

Experiment 2:

Based on the predicted lithium concentration in the muscle tissues after feeding a high dose of LiCl (450 mg/kg), lithium concentration peaked at approximately 100 hours post-ingestion (Figure 2). Lithium concentrations slowly declined thereafter, and never reached basal level by the end of the experiment (240 hours). A high mortality rate at this dosage was observed (12 out of 14 total treated, ~86% mortality) with most of the mortalities occurring after 73 hours post-ingestion. Approximate death times post ingestion of 450 mg/kg LiCl were as follows; 48h - 1 dead; 73h - 2 dead; 97h - 2 dead; 145h - 2 dead; 169h - 5 dead. Kidney, liver, and muscle tissue samples were obtained from all the animals that died. Behavioral observations were once again recorded for treated animals following LiCl ingestion. Treated animals appeared unaffected until 24 hours post-ingestion when they stopped eating, drinking, and moving around the containment area. Because of the high mortality rate, we were unable to construct a complete depletion curve for this concentration.

Experiment 3:

Mean lithium concentrations were different among muscle, kidney, and liver tissues within 7 hours after ingestion. Mean lithium concentrations in both liver and kidney tissues were greater than muscle at 7 and 24 hours post ingestion (P < 0.01; Table 1). There was no difference in lithium concentrations between the three tissues at 96 hours (P > 0.05) (Table 1). Mean lithium concentrations remained elevated (P < 0.01) in both liver and kidney in the first 24 hours after LiCl ingestion but returned to basal level at 96 hours after ingestion. Although, the overall concentration of lithium was less in muscle tissue, lithium concentrations remained elevated (P < 0.01) in muscle in the first 24 hours after LiCl ingestion and returned to basal level at 96 hours after ingestion (Table 1). There was not a tissue type by time interaction effect on Lithium concentrations.

^a,b Means with different superscripts within a column differ (P< 0.05)

^x,y Means with different superscripts within a row differ (P<0.05)

A 150 mg LiCl/kg body weight was selected as the low dose based on previous reports of effectiveness in creating taste aversion in domestic sheep, cattle, and caribou [9,22,23]. Administering LiCl dosages greater than 300 mg/kg body weight is rare within the literature, and an exact toxic dosage in small ruminants has yet to be determined. The LiCl toxicity in mice occurred at a 600 mg LiCl/kg body weight [24], and to avoid exceeding the toxic threshold for ruminants the high dosage was reduced to 450 mg LiCl/kg body weight in the current study. However, with the multiple mortalities occurring post-ingestion the toxic threshold apparently was exceeded in the sheep indicating the LiCl toxic threshold maybe even less than 450 mg LiCl/kg body weight.

Maximum lithium concentration levels and withdrawal periods within muscle tissue may vary by dosage, and among animals to an extent. Most notably in experiment 1, one sheep at 96 hours had a greater Lithium concentration (9.8 μg/g) causing a larger variation in the data at that time point (Figure 1). Interestingly, mean muscle lithium concentration at 96 hours post ingestion was similar to the pre-ingestion concentration when the data from that sheep was not included in the data analysis. As indicated, at the low dosage, lithium concentration increased within muscle tissue starting with the first biopsy samples taken at 4 hours and continued to increase until the maximum concentration value occurred at approximately 24 hours. Following the peak, lithium concentrations quickly declined and returned to basal levels by 192 hours. (Figure 1) Although lithium in kidney and liver samples did not return to baseline concentrations from the low dose, at 96 hours post-ingestion, there was not a difference between lithium concentrations among the three different tissues suggesting most of the lithium had been metabolized and excreted leaving behind small residual amounts in all tissues. These results are similar to withdrawal periods of lithium in different types of excreta in sheep and goats previously reported [18].

In this study feed and water intake pre- and post-ingestion were not directly quantified, but treated animals were observed for behavioral changes. Although previous studies have observed signs of malaise (head droop and inactivity) [21] and an aversion to food post LiCl ingestion [7,15], we did not observe either of these behavior changes. Treated sheep were consuming provided alfalfa immediately following LiCl drenching and continued to do so throughout the study period. The low dose LiCl may not have been high enough to produce the taste aversion in sheep, and perhaps a greater dose of LiCl (200-300 mg/kg) may produce the taste aversion without producing the toxic effects seen at a LiCl dose of 450 mg/kg.

Only 2 of the 14 individuals that received high dosage (450 mg/kg BW) did not succumb to toxicity, and after 240 hours postingestion muscle tissue samples from the surviving animals had yet to reach basal level. Thus, a complete withdrawal time for a dosage of 450 mg LiCl/kg body weight was not determined. Despite supportive treatment for dehydration animals succumbed within a few hours of clinical signs. As indicated, majority of the mortalities occurred between 36- and 193-hours post-ingestion. Multiple symptoms of toxicity were observed including lack of appetite, malaise, severe dehydration, hypoglycemia, muscular tremors, increased heart rate, and extreme diarrhea. Necropsies were conducted by a veterinarian, and cause of death was determined for each deceased animals. In the absence of any additional postmortem findings, it was determined that all animals had died due to LiCl overdose, and that 450 mg LiCl/kg body weight appears to be a lethal dose for small ruminants.

Although treated animals only received a single dosage of LiCl, the high-level potency of the chemical compound resulted in death as the physiological responses in the body, and especially the kidneys, were not able to process and excrete excess LiCl resulting in accumulation and eventual death [17,25]. Kidneys are the main processing organ that excretes LiCl [17,25], and excess lithium can disrupt the absorption of salt and water, often leading to polyuria [26]. If the kidneys are not able to process and excrete the ingested amount of lithium, excess amounts begin to accumulate in other tissues [17]. This is likely what occurred in the high dose trial and why our results show no difference in lithium concentrations among the tissue types. Once lithium levels in the kidney exceeded maximum intake, surplus lithium may have deposited in the liver and muscle tissues, resulting in all 3 tissue types containing high concentration levels. However, in the low dose, the highest lithium concentrations were in the kidneys, followed by liver, and the least amount of lithium concentration was in muscle tissue. This was likely because of the kidneys being able to function correctly with a manageable intake of lithium. Overdosing was not an issue as the amount of ingested lithium was processed and excreted by the kidneys without excess accumulation.

Conclusion

The muscle concentration of lithium at a low dose of 150 mg/ kg body weight of LiCl administration reached baseline lithium in muscle tissues by 192 hours post-ingestion. Although, the withdrawal period within the liver and kidney for this dosage was not established, the lack of difference in lithium concentration between the three tissues (muscle, liver, kidney) at 96 hours suggests lithium concentrations of liver and kidney would not differ from baseline by 192 hours. Likewise, high dose withdrawal periods for all 3 tissue types were undetermined due to 450 mg LiCl/kg body weight being lethal for many sheep. It appeared that kidney tissues retain the greatest amount of lithium, followed by liver tissues, and lastly muscle tissues. It is important to acknowledge the toxic threshold for domestic sheep, and likely for other small ruminants, lies between 150-450 mg LiCl/kg body weight.

Although we didn’t notice any instant food aversion after ingesting LiCl, this chemical could be a useful deterrent for lowering WTD crop destruction. Although sheep and deer have similar body sizes and rumen capacities, it should be noted that toxicity effects and withdrawal times for each tissue type may differ between species. Based on the results of the current study, a 192-hour withdrawal period in muscle tissue for a low dose of LiCl in domestic sheep may be taken into consideration; however, the analyses for other tissue types at low dosages and for all tissue types at high dosages were inconclusive, hence withdrawal period cannot be recommended.

Therefore, we suggest that before field implementation and human consumption of an animal that has ingested LiCl, more trials are necessary that include using LiCl at a dose range of 200 - 300 mg/kg for longer time periods, with larger samples sizes, and incorporate a variety of ruminant species.

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