Reports or descriptions of medical management of thermal injuries in donkeys is lacking. Four donkeys sustained burn injuries during the wildland–urban interface fire in Valparaiso, Chile, in 2014. The donkeys received first aid care at the scene of the fire, and then were hospitalized and treated for three months for thermal injuries, of various degrees of severity, in several body parts. The clinical findings and treatment of four of the donkeys are described in this paper. All donkeys recovered and were retired to an animal shelter.Burn injuries are uncommon in large animals and there are no reports of these injuries in donkeys. Burns cause local and systemic effects. Extensive thermal injuries can be challenging to manage and the extent of the burn surface affected will directly impact the severity of the illness and the prognosis. Burns are classified according to the depth of injury into four categories, from first-degree burns, and the least affect to fourth-degree burns, which are the more severely affected patients. This case report describes the medical management of four donkeys that sustained various degrees of external burn injuries during the wildland–urban interface fire in Valparaiso, Chile. The donkeys were treated topically for several weeks and closely monitor for inadequate nutritional intake. Water based topical medications are preferred in burn cases because they can be easily applied and removed without interfering with wound healing. Of note, the caloric demands of these cases can be achieved by increasing the amount of grain, adding fat , and free-choice alfalfa hay. All donkeys recovered and were retired to an animal shelter. Burns are damage to body tissues caused by the action of high temperature or hot solutions, lightning, friction, abrasions, or certain chemicals  . Burns aFrom 12 to 14 April 2014, a wildland-urban interface (WUI) fire occurred at a catastrophic scale. Over 2827 acres were burned, 11,000 people affected, and 2900 homes destroyed in Valparaiso, Chile. WUI fires occur more frequently during periods of drought and climate change. The wildfires also significantly affect land used for agriculture and wildlife habitats, injuring and killing large numbers of wildlife and farm animals. Valparaiso has a peculiar topography characterized by 45 hills, and the risk for wildfires exist in most parts of rural areas every summer. The city of Valparaiso was declared a World Heritage Site by United Nations Educational, Scientific and Cultural Organization (UNESCO) in 2003. The estimated number of affected domestic animals associated with the WUI fire was 146 , of which many of them died. Many of those animals were examined and treated by veterinarians for burns and/or smoke inhalation. Four donkeys suffered burns, two of them severe, requiring long-term hospitalization, and costly medical treatment, therefore, the owner donated the donkeys to the Veterinary Clinic Service of Viña del Mar University.During fires, thermal injuries are caused by direct exposition to the flames and/or inhalation of toxic gases . InhalatClassification of the burn injuries is considered one of the more important determinants of outcome. First-degree burns affect only superficial layers of the epidermis, causing erythema, edema, and desquamation, and they heal without complication. Second-degree burns involve the superficial and/or deep portion of the epidermis. Superficial second-degree burns affect the stratum corneum, stratum granulosum, and a few cells of the basal layer; however, deeper burns involve the epidermis, including the basal layers. Such burns are characterized by erythema and edema at the epidermal-dermal junction, necrosis of the epidermis, accumulation of leukocytes at the basal layer of the burn, eschar formation, and minimal pain . These mIn addition to the depth of the burn, the extent or total body surface area (TBSA) affected by thermal injuries also needs to be assess. The “Wallace Rule of Nines” system used in human medicine estimates the prognosis according to the extent of the burn. This system divides the body surface area into regions that represent multiples of nine and allocates body regions as follows: each limb, head, neck, thorax, and abdomen represent 9% . SpecifiDonkeys are particularly susceptible to hyperlipemia in stressful circumstances. It can progress rapidly and is often life-threatening. Prompt diagnosis and treatment is required to improve the outcome. If managed correctly, with an appropriate diet and regular (routine) preventive healthcare, this risk can be minimized .This report describes the medical management of veterinary care of thermal injuries in four donkeys.Case 1 (D1): a 7-year-old male donkey, weighing 150 kg was examined and treated within 2 h after being burned. The donkey was kept in a small pen (10 × 10 m) with three other donkeys. Upon physical examination, the body condition score (BCS) was 3/5, tachycardic 64 beats/min (reference range 36–52), tachypneic 30 breath/min (reference range 12–28), furthermore, the donkey had congested mucous membranes. There were burn marks over the lips, face, ears, dorsum, and both front limbs. All serous exudate was observed over the coronary bands in front limbs, but had a normal gait. Immediate treatment included flunixin meglumine 1.1 mg/kg intravenously (IV) ; hosing with tap water (13 o 15°C) all over the body for 30 min to protect the tissue, and bandage with sterilized gauze and liquid paraffin gauze dressing over the neck and back prior to transportation to the clinic 2 h later A.Approximately 12 h after the incident, the estimated TBSA was approximately 40–50%. Most of the affected areas were classified as deep second-degree burns with some small areas of third-degree burns on the distal area of the front limbs and face. There was also facial, abdominal, and limb edema. The donkey was eating and drinking with difficulty (dysphagia—possibly caused by edema and pain). He was placed on IV fluids with Hartmann’s solution 10 mL/kg bwt/h  for 3 h until he was stable and started to eat and drink. Both eyes had bilateral corneal ulcers and uveitis B, with sA topical cream of sulfanilamide with zinc oxide was applied to the coronary bands .Respiratory distress was observed, characterized by tachypnea, nostril flaring, and cough—a suspected result from smoke inhalation; therefore, treatment with corticosteroid, dexamethasone sodium phosphate 0.2 mg/kg IV  was administered. A parasympatholytic drug and beta-2 agonist inhalation therapy by aerochamber , using ipratropium bromide 0.8 µg/kg q 6 h  and albuterol 2 µg/kg q 4 h  were also administered. The aerochamber was placed in one nostril and the opposite side was occluded . Airway Complete blood cell count and serum biochemistry profile were performed on the second and eight days post rescue . The first analysis, done at day two, revealed: neutrophilia, increased alkaline phosphatase, muscle enzymes creatine kinase, aspartate aminotransferase and triglycerides, decreased total protein and albumin, increased glucose, and decreased total calcium and chloride concentration. Slightly cloudy serum was observed in the clotted blood sample .The second analysis at day 8 showed increased alkaline phosphatase, aspartate aminotransferase. Blood analysis was repeated on day 15 of the hospitalization and all of the parameters were within normal limits . AntibioDuring the hospitalization time period (90 days), the burn surfaces, appetite, and pain level of the donkey were monitored. The donkey appeared more comfortable, with good appetite, and showed interest in socializing with other donkeys and his environment; these responses became evident since day 7 in the clinic. The edema (described) was observed since the first day of the rescue and decreased after 6 days, the skin became dry and sensitive to touch, and he experienced hair loss on his legs, neck, and back.Yucca schidigera extract 954 mg + vitamin B12 10 mcg/ 10 mL, Bute-Less Paste, Absorbine, Massachusetts, MA, USA), 5 mL oraly q 24 h/15 days, supplement to discomfort and pain relief. Omeprazole 2 mg/kg per os q 24 h/4 weeks  was given for treatment and/or prevention of gastric ulceration.Flunixin was given daily for pain control for 10 days since the rescue , and continued with a natural product  as potent analgesic for the first six days in the clinic. On day seven, sedation and potent analgesic for treatment was no longer required. Local anesthetic instillation with lidocaine hydrochloride was used in some affected areas on the back and face .Buddleja globosa , raw honey, aloe vera, and nitrofurazone cream . Nitrofurazone was used in areas with exudation and erythema and, locally, raw honey on areas with less exudation and erythema. Dry skin areas were treated with Jelonet, and then it was changed to Buddleja globosa cream. Fly repellent was used around wounds .Wounds were lavaged every 24–36 h, for debridement of devitalized tissue, and dead skin was removed with sterile scissors or a hypodermic needle. The blisters were left intact, however, gauze dressing with impregnated paraffin (Jelonet) was used in some areas where blisters opened. Local antibacterial drugs were used every 24 h. The wounds were lavaged with chlorhexidine gluconate solution  and diluted in water with sterilized gauze using gloves and triclosan . A variety of topical products were used in conjunction with the dressings for the following weeks: cream of Three weeks later, the donkey developed pruritus, which was treated with chlorpheniramine maleate 0.5 mg/kg IV  as needed.After 2 months, a thin layer of skin started to regrow. Exuberant granulation tissue on some areas was periodically de-bulked. After 3 months, all treatments were discontinued, and most of the burned areas had healed, but the area of the face and front limbs were without hair growth. At this time, the only product used on his legs and body was aloe vera gel. His coronary bands from the front limbs did not separate significantly, but a horizontal line of damage was evident around the hoof wall (1 cm below the coronary band), walk, and trot sound.Case 2 (D2): a 15-year-old female donkey, 120 kg, was examined with D1. The jenny appeared anxious and painful. Upon physical examination, her BCS was 3/5, she was tachycardic (72 beats/min), tachypneic (30 breath/min), and had congested mucous membranes and muscle tremors. Burns over her face, neck, back, perineal area, vulva, and coronary band swelling of the left hind limb were observed. She was transported to the clinic with the other affected animals and was treated, as per case 1.Her burns were classified as second-degree, with some areas with third-degree ; estimated TBSA approximately 40–50%. She had bilateral corneal ulcers and uveitis, and her eyes were treated with the same antibiotic ointments and regime as case 1. The left coronary band was inflamed and a serious exudate was present; therefore, topic cream with sulfanilamide + zinc oxide was applied. She was five-months in gestation, which was closely monitored at the clinic. Intravenous fluids with Hartmann’s solution were administered at the same rate as described above, as well as flunixin and omeprazole. Fluids were discontinued on the second day at the clinic, when the laboratory report indicated adequate hydration. Moreover, she was eating hay with a good appetite at this time.Complete blood cell count and serum biochemistry profile were performed in the clinic similar to D1. The first blood analysis, taken at day two, revealed: neutrophilia, increased alkaline phosphatase, muscle enzymes creatine kinase, aspartate aminotransferase, triglycerides and cholesterol, decreased total protein and albumin, increased glucose, and decreased mineral calcium and chloride concentration. Slightly cloudy serum was observed in the clotted blood sample .Antibiotic therapy started with procaine penicillin + dihydrostreptomycin IM, and for the skin, aloe vera cream, at the same doses rates described in D1. The wounds from the skin were periodically lavaged and cleaned using disinfectant, and a similar topic cream, as described in D1, removing the devitalized tissue when necessary . The pinna of the left ear also developed ulcers and permanent cartilage contracture, as D1. A second blood analysis performed eight days post rescue showed increases in alkaline phosphatase and aspartate aminotransferase. Concerning the third blood analysis performed on the 15th day of hospitalization, all parameters were within normal limits . PrurituThe left coronary band lesion, increasing in size and lameness, was graded 2/5. Radiographs were taken, which revealed no sign of distal phalangeal rotation. Flunixin meglumine (1.1 mg/kg) for pain was continued, daily, for 12 days , and the supplement for pain relief was the same dose rates as D1. After six weeks, she could canter when turned out into a paddock in the evenings, or early mornings, when the outdoor temperatures were cooler. His left coronary band from the hind limb did not separate, but a horizontal line of damage was evident around the hoof wall (1 cm below the coronary band).The jenny was discharged after 3 months of hospitalization. She was doing well on her 3- and 8-month pregnancy checks. She foaled a healthy donkey foal after 13 months of gestation (range of 11–14.5 months) [Cases 3 and 4 (D3–D4): the next two cases were a 12-year-old female donkey, 160 kg, and a 5-year-old male, 110 kg. Both were examined at the site of the fire, as described for both previous cases (D1 and D2). They both had a BCS of 2/5, were tachycardic, and small burn areas with hair loss on their heads (noses and cheeks), as well as necks, were observed. Their TBSAs were approximately 5% and the burn areas classified as first degree. These two donkeys were in the same paddock with the others; however, they were less affected. Cases D1–D2 were tied up to a fence, close to the burning vegetation until the rope was burned by fire, while cases 3 and 4 were free. This most likely explains the severity of the injuries in the first two cases.D3 and D4 were also hosed with tap water all over their bodies, and moved to the same clinic. They were also treated with flunixin meglumine for 3 days and the burn areas were treated with aloe vera as the previous two cases. Superficial burns healed without complication. The areas affected became dry between 5 and 8 days. The wounds from the skin were periodically lavaged and cleaned using disinfectant, and similar topic cream, as described in D1, was used. It was necessary to use xylazine and butorphanol tartrate for wound management for 2 days after they arrived at the clinic. After 3 weeks, most of the burned areas healed .The hematologic and biochemical analyses of D3–D4 performed two days after the recue showed no abnormalities . They shMedicago sativa and Lolium perenne), 1.7% daily of their bwt in dry matter (DM) intake, which is equivalent to 2.6 kg D1, 2.1 kg D2, 2.7 kg D3, 1.9 kg D4 per day , continuous wet dressings (semi-open technique), eschar formation (exposed technique), or excision and grafting . In thisCleaning with mild soap and water was used to facilitate softening and removal of dead skin. Aloe vera appeared to be helpful for managing dry skin, which appeared to prevent the skin from sloughing, and allegedly has anti-inflammatory effects and promotes wound healing ,26. ThisHoney has been used in burn patients for many years. The antibacterial activity of honey, its low pH, high viscosity, hygroscopic effect, and hydrogen peroxide content, may play a combined role in its effectiveness for the treatment of burns, and may also provide a moist environment for optimum healing conditions ,29. TherThe donkeys with corneal ulcers (D1 and D2) responded well to treatment and healed with no complications. Human studies report 7.7%−18% of the incidence of chemical and thermal injuries to the eye of all ocular trauma, with corneal ulcers and blepharospasm . Burned Long-term use of nonsteroidal anti-inflammatory drugs (NSAIDs) did not result in adverse effects during hospitalization, but other NSAIDs could have been used described for donkeys ,31. GastThe application of sulfanilamide with zinc oxide around the coronary band was an effective topical treatment in these cases. Sulfanilamide acted with a differential bactericidal activity, and mild astringent, and decongestive effect, in addition to the absorbent and insulating properties conferred by zinc oxide ,33. CoroPreventive antibiotic therapy was used in D1 and D2, but the literature mentioned that the role of prophylactic antibiotics for severe burns is controversial, both in humans and horses . TherefoA hallmark of burn injury is a hypermetabolic response that results in significant pathological alterations in a number of tissues . StudiesIn veterinary emergency medicine and critical care, teamwork to provide adequate patient care is essential. The entire team consisted of two veterinarians, a technician, and eight graduate students to provide continues care. The care and devotion provided to each animal by the veterinarian staff highlights the concept of the animal-human bond and the dedication veterinarians provide to their patients.Domestic and wild animals can suffer major thermal injuries during wildfires, and reports of these injuries in donkeys is lacking. This manuscript describes the clinical findings, medical treatments, and evolution of four donkeys that suffered thermal burns. All of the donkeys recovered and were sent to an animal shelter after their recovery.


Bixa orellana is a plant from the Bixaceae family, for which, limited information is available on hepatoprotective properties. This study aimed at evaluating the protective effects of this plant on sub-acute acetaminophen (APAP)-induced liver injury in mice. Various aqueous extracts were prepared from roots, leaves, and barks. Albino mice were divided into six groups: a control group, an APAP group; a silymarin group (positive control) and three test groups. Mice were treated orally with APAP (250 mg/kg) followed 3 hr later by plant extracts, silymarin (50 mg/kg) or distilled water (10 ml/kg) administration once daily, for seven days. After treatment, animals were sacrificed, the liver was collected and different biochemical parameters were measured. Histological analyses were performed using hematoxylin/eosin staining and the qualitative phytochemical content of plant extracts was evaluated using conventional methods. B. orellana barks decoction (250 mg/kg) significantly reduced alanine aminotransferase levels (p<0.001), unlike leaves and roots extracts. Moreover, the bark infusion had the highest activity compared to macerate and decoction. It significantly reduced malondialdehyde levels (p<0.001) and increased the levels of glutathione, superoxide dismutase and catalase, at doses of 250 and 500 mg/kg compared to the APAP group. A significant (p<0.001) decrease of tumor necrosis factor-α levels and leukocyte infiltration was found following treatment with bark infusion. The infusion content evaluation revealed the presence of polyphenols, saponins, tannins, sterols, anthraquinones, and coumarins and the absence of alkaloids. Administration of B. orellana barks is hepatoprotective against APAP-induced toxicity via antioxidant and anti-inflammatory mechanisms.These results show that infusion from  The liver is the largest internal organ in vertebrates and some other animals. It controls and regulates a wide variety of biochemical reactions including the biotransformation and elimination of xenobiotics, making it more sensitive to the toxicity of these agents , dissolved in distilled water and administered to animals at a dose of 50 mg/kg. The hepatotoxin used was APAP tablet (Doliprane 500 mg). These drugs (APAP and silymarin) manufactured by Sanofi-Aventis  and Micro Labs Limited  respectively, were purchased at the Pharmacy of SARE, in Ngaoundéré, Cameroon. The alanine aminotransferase (ALT) and tumor necrosis factor alpha (TNF-α) kits used for the assays were from Linear Chemicals .S.L. Joaquim Costa 18 2a planta  and Solarbio Life Sciences , respectively.Plant materialB. orellana, harvested on the campus of the University of Ngaoundéré, in the Vina Division of the Adamawa Region of Cameroon. Samples were collected on August 10, 2018, very early in the morning between 6 and 10 am. The botanical identification of the plant was made by Prof. Mapongmetsem Pierre Marie, a botanist at the Faculty of Sciences of the University of Ngaoundéré, Cameroon. After harvest, plant samples were washed with tap water to remove dirt, cut into small pieces to facilitate drying, dried in the shade for 3 weeks, then crushed and sieved through a mesh of pore size 695 μm in diameter, to obtain powders.The plant material consisted of the roots, leaves and barks of Preparation of aqueous extractsB. orellana bark powder was weighed and mixed with 250 ml of distilled water. For preparing the decoction, the mixture was boiled at 100°C for 20 min. For the infusion, powder was mixed with boiling water for 20 min, while for macerate, the powder was just mixed with distilled water for 20 min at room temperature. After cooling, the mixtures were filtered and the filtrates were frozen and lyophilized at -55°C. A yield of 4.68% was obtained for the decoction, 5.92% for the infusion and 6.28% for the macerate. The freeze-dried extracts were packed in aluminum foil and stored in boxes at 4°C until use. Twenty-five (25) g of the Phytochemical analysesPlant samples were analyzed using conventional methods to determine the existence of alkaloids, flavonoids, polyphenols, tannins, saponins, coumarins and anthraquinones, as previously reported  aged 2 to 3 months, weighing 18 to 35 g, were used. Animals were provided by the pet store of the National School of Agro-Industrial Sciences of the University of Ngaoundéré (Cameroon) and placed in plexiglass cages containing chips, provided with a steel grid preventing their exit. Mice had free access to food and tap water and were kept at room temperature under conditions of the animal facility. Mice were used according to the Animal Research: Reporting in vivo Experiments (ARRIVE) guidelines and permission for experiments was given by the Animal Ethics Committee of the Faculty of Science of the University of Ngaoundéré (200/2019/UN/DFS/VD-RC/CSPDR).Albino mice  mice were divided into six groups of 5 mice as follows: Group I: normal control (NC) received 10 ml/kg of distilled water; Group II: negative control (APAP) treated with 250 mg/kg of APAP Lee, 2017; Group Ith day, animals were sacrificed and the livers were collected, crushed with a mortar and pestle and centrifuged at 4000 rpm for 10 min at 4 ° C. ALT levels were then measured in liver samples. At the end of this test, the method of preparation, having shown the most effective hepatoprotective effect, was retained for further investigations. At the end of this experiment, the most active extract was identified and different modes of its preparation were tested to determine the most efficient one. Then, mice were grouped and treated as mentioned above. However, in Group IV, the three batches received 250 mg/kg of APAP and 250 mg/kg of decoction, macerate or infusion from the most effective plant part. Treatments lasted 7 days and on the 8per os respectively to each batch. The fourth batch received only 500 mg/kg of bark’s infusion and no APAP, to test the extract toxicity. All four batches were treated daily for one week. Thereafter, animals were sacrificed, the liver homogenates were prepared and the biochemical parameters were measured. Seven batches of 5 mice each, were used in this third test as follows: Groups I, II and III were treated as mentioned above. Group IV was divided into four batches. The first three received 250 mg/kg of APAP and 3 hr later, 100, 250 and 500 mg/kg of the bark’s infusion was administrated  Preparation of liver tissue homogenatesUpon dissection, liver samples were collected for the preparation of liver homogenates. A portion of the liver was ground in a mortar, then 1 ml of the ground material was homogenized in 9 ml (1/10) volumes of 0.1 M phosphate buffer, pH 7.4. The homogenate was centrifuged at 10,000×g for 15 min at -4°C and aliquots of supernatant were used for biochemical estimations.Assessment of markers of liver functionALT levels were determined as previously described  and superoxide dismutase (SOD) were assessed as antioxidant parameters using commercial detection kits  according to the manufacturer's instructions. The degree of lipid peroxidation was measured by quantifying malondialdehyde (MDA) levels as previously reported .Histopathological examinationThe liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned into 5 μm thickness, and stained with hematoxylin and eosin (H&E) for evaluation of histopathological changes. Microscopic observations were performed under a bright-field microscope. Different pathological grades were considered in order to evaluate the severity of inflammation. Grade 0 , grade 1 , grade 2 (mild), grade 3 (moderate), grade 4 (important leukocyte infiltration)  followed by a Dunnett Multiple Comparisons test, using GraphPad prism5.0 software. A difference was considered statistically significant at p<0.05.Effect of different parts of Bixa orellana on APAP-induced liver injury B. orellana on ALT levels in APAP-induced hepatotoxicity. According to this Figure, the bark and leaf extracts at 250 mg/kg exhibited a significant (p<0.001 and p<0.05 respectively) reduction in ALT levels while the reduction induced by the root extract was not significant. Of these 3 extracts, the bark had the most pronounced reduction in ALT levels, similar to that of silymarin used as the positive control.Effect of the mode of preparation of Bixa orellana bark extracts on ALT levelsEffect of bark’s infusion on ALT levels B. orellana on ALT levels in the liver of intoxicated mice. All the tested doses significantly alleviated ALT levels compared to the negative control (p<0.05 and p<0.01). Similarly, a significant decrease was found (p<0.01) in the positive control compared to the negative control. For the groups of mice that received only plant extract at 500 mg/kg, there was no significant difference compared to the normal control, suggesting the safety of this plant extract.Effect of the bark’s infusion on oxidative stress markersB. orellana inhibits lipid peroxidation in the liver. The effect of bark’s infusion of B. orellana on the activity of CAT and SOD was also evaluated (valuated . In APAPEffect of the bark’s infusion of Bixa orellana on TNF-α expressionInflammation and oxidative stress are two important processes during APAP-induced hepatitis. Since bark’s infusion was found to interfere with oxidative stress, we investigated its effects on the pro-inflammatory mediator, TNF-α. Effects of the bark’s infusion of Bixa orellana on histological sectionsTo confirm the necroinflammatory activity on liver tissue, the effect of bark’s infusion was also examined by H&E staining. As shown in Phytochemical profileThe qualitative phytochemical analysis of the different aqueous extracts showed some differences in the composition of macerate, infusion, and bark decoction. The infusion contained most of the metabolites tested, except alkaloids, while only saponins were missing from the bark macerate, and flavonoids and alkaloids were absent in bark decoction .The liver is an essential organ in many biological functions including the metabolism of carbohydrates, lipids, and proteins, the regulation of blood clotting, the cholesterol transportation, resistance against infections, in addition to metabolism and clearance of xenobiotics (Fortner and Blumgart, 2001Bixa orellana, a plant of the family of Bixaceae, was reported to possess protective activities against carbon tetrachloride- and ethanol-induced hepatotoxicity, but the effects on APAP were poorly studied. In this work, we evaluated in vivo hepatoprotective activities of different aqueous extracts of this plant on APAP-induced liver injury. A first screening of the bioactivity of roots, leaves, and barks aqueous extracts showed a more pronounced inhibition with the bark extract, suggesting, a concentration of active metabolites in this plant part compared to other parts. Further investigations with aqueous extracts from the bark revealed a significant effect with the infusion compared to decoction and macerate, demonstrated by the decrease of ALT levels. This result might indicate that heat played a role in the extraction process of active ingredients as reported previously (Chavan et al., 2001Solanum fastigiatum hepatoprotective activities to the presence of flavonoids in the water extract (Sabir and Rocha, 2008B. orellana in the rat liver (Bell et al., 2012APAP is the most commonly used analgesic and antipyretic medication. Taking this drug at excessive doses generates a reactive compound NAPQI, that may accumulate in the liver and therefore lead to antioxidant depletion or trigger hepatic cytolysis (Hinson et al., 2010B. orellana has been shown in this study as an extract with anti-inflammatory and antioxidant potential in APAP-induced liver damage.The mechanisms of APAP hepato-toxicity are well described, and the role of inflammation in initiating and propagating liver injury was highlighted by evidence (Sabir and Rocha, 2008B. orellana is more active than leaves and roots extracts. Moreover, the bark’s infusion of B. orellana was a more potent hepatoprotective extract than macerate and decoction, acting mainly on sub-acute APAP liver injury through anti-inflammatory and antioxidant mechanisms. In addition, the activity was comparable with that of silymarin. This work paved the way through the isolation of active ingredients from bark infusion, which will be advantageous to produce new bioactive compounds with greater activity against liver damage. Extract from bark of


Postpartum hemorrhage (PPH) is the leading cause of maternal morbidity and death worldwide. The history of cesarean section and the occurrence of placenta previa were significantly associated with the increase in blood transfusion. Therefore, to prevent PPH, it is important to understand the effect of blood transfusion during cesarean section on postpartum hemorrhage. The purpose of this study is to determine the cause of blood transfusion during cesarean section, especially large amounts of blood transfusion, and to take measures to reduce the blood demand caused by PPH with limited blood supply.This study was a retrospective study of patients who underwent blood transfusion during cesarean section in Qilu Hospital of Shandong University (China) from January 2013 to December 2016. Red blood cell infusion ≥10 U during cesarean section was defined as massive blood transfusion. The study collected the demographics of pregnant women, obstetric characteristics and reasons for blood transfusions, as well as blood components and blood transfusion results. Multivariate regression analysis was performed for evaluating the risk factors of PPN.P < .001). The history of cesarean section (P = .005) and the occurrence of placenta previa were positively correlated with the increase in blood transfusion (P = .016). There were 72 cases of massive blood transfusion, accounting for 12.27% of blood transfusion patients. Among mass blood transfusions, 93.1% of cases had prior cesarean delivery, and placenta previa accounted for 95.8%. 19.4% of patients receiving massive blood transfusions underwent hysterectomy. There was no significant difference in maternal BMI and gestational age between the mass blood transfusion group and the non-mass blood transfusion group.From 2013 to 2016, a total of 587 patients received blood transfusions during cesarean section. The proportion of women receiving blood transfusion during cesarean section increased (from 3.21% to 7.40%, From 2013 to 2016, the demand for blood transfusion, especially the demand for massive blood transfusion, increased. Repeated cesarean section and placental previa combined with uterine scar are positively correlated with increased blood transfusion. Reducing the initial cesarean section should help reduce the massive blood transfusion caused by placenta previa with a history of cesarean section. Importantly, the PPH rate is increased from 1.5% in 1999 to 4.1% in 2009 in Iraland. The risk factors for PPH include multiparous, obesity, repeated cesarean section, advanced maternal age, and pregnancy comorbidities such as pregnancy-induced hypertension.–7Postpartum hemorrhage (PPH) is a leading cause of maternal mortality. All pregnant women with gestational age of >20 weeks are at risk of PPH.–10 Of note, intraoperative bleeding of pernicious placenta previa is rapid and massive, hemostasis is difficult, and large amounts of blood transfusion and sometimes hysterectomy are necessary to control bleeding. Moreover, PPH associated with pernicious placenta previa disease is extremely dangerous and may lead to serious complications such as hemorrhagic shock, disseminated intravascular coagulation (DIC) and metabolic acid-base disorder.,12In China, family planning and one-child policy has been implemented for more than thirty years to control population growth, with some exceptions. As the population aging problem has intensified, each couple has been allowed to have 2 children since the end of 2013. Nevertheless, with the introduction of this policy, the increasing in births has led to blooming in the demand for obstetric services, as well as repeated pregnancy and incidence of pernicious placenta previa.Increasing the need for massive blood transfusion has been noticed clinically with impact of two-child policy in China. Some risk factors are associated with PPH, such as massive blood transfusion during cesarean section, pernicious placenta previa and combined gestational hypertension. Altering those modifiable factors might be able to reduce transfusions and PPH. The hypothesis of the study is that the previous cesarean delivery and uterine scar are associated with the demand of blood transfusion. The aim of our study was to analyze the changes in blood transfusion during cesarean section, and advocate the reduction of primary cesarean section to decrease adverse consequences caused by the history of cesarean section.22.1China (No: 2017018).This was a retrospective study of patients who received blood transfusion during cesarean section in Qilu Hospital of Shandong University between January 2013 and December 2016. The study was approved by the Ethics Committee of Qilu Hospital of Shandong University, ≥10 U (1 U of red blood cell suspension was prepared by 200 ml of whole blood) was considered to be massive blood transfusion.Data was extracted from digital medical record management system of Qilu Hospital of Shandong University. According to clinical cases of blood transfusion during cesarean section, national version of the International Classification of Diseases (ICD)-10 diagnosis coding and Beijing version of the ICD-9 surgery code were used to confirm obstetric comorbidities. The inclusion criteria included infusion of any blood products during cesarean section, such as red blood cell, plasma, platelets and cryoprecipitate. An amount of red blood cell infusion 2.2≥10 U, and indications for hysterectomy. The estimated blood loss based on surgical records was established from the suction device, gauze pad and vaginal bleeding.The collected data included age of patients, gestational age, body mass index (BMI), gravidity, singleton/multiple pregnancy, history of hypertension, obstetrical pathologies, any other significant comorbidities, number of previous abortions, history of cesarean section or uterine surgery, clinical diagnosis, surgical time, anesthesia type, hospitalization, postoperative hospitalization, emergency vs. elective surgery, neonatal outcomes, intraoperative bleeding volume, infusion of erythrocytes, plasma, platelets and cryoprecipitate, preoperative and postoperative hemoglobin, platelets, prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen, patients with red blood cell infusion 2.3P value <.05 was considered statistically significant.Sample size analysis was carried out by one way ANOVA – power analysis was used to achieve a power of 0.80 at a α level of 0.05. Statistical analysis was conducted using SPSS 23.0 . Continuous variables with normal distribution were presented as mean (standard deviation [SD]); and were analyzed using the ANOVA followed by Bonferronis post-hoc comparisons tests. Categorical data were presented as frequencies and were analyzed using the chi-square test. Two-sided 33.1P < .001). The gravidity and parity numbers and previous cesarean section rate had significant differences from P = .02 and P = .006 from 2013 to 2016 . The blood transfusion rate due to preeclampsia was high in 2013, but decreased in 2014, 2015, and 2016 (P = .02). The frequency of blood transfusion during cesarean section in patients with placenta previa increased from 29.69% in 2013 to 51.10% in 2016 (P = .02). Moreover, the proportion of patients with pernicious placenta previa among patients who received blood transfusion increased constantly from 9.38% to 32.6% (P < .001).The common causes of blood transfusion during cesarean section included placenta previa, anemia, preeclampsia, placental abruption, acute fatty liver of pregnancy (AFLP), and other blood diseases Table . During 3.3>10 U of red blood cells were tend to be elderly maternal, older (P = .001), or a higher gravidities and garities (P < .001), or a higher incidence of previous cesarean section (P < .001), or a lower occurrence of gestational hypertension (P < .001), or a higher incidence of placenta previa (P < .001), or a higher incidence of hysterectomy (P < .001), and or a higher incidence of general anesthesia (P = .004). The stratification of different age groups was classified as: <24 years old young women, 30 to 39 years old women and women ≥40 years old. However, according to the maternal age proposed by RCOG and ACOG, the evidence has not been confirmed, and the reason may not be reasonable. Compared to multiple pregnancy, singleton pregnancy showed a significantly higher rate for receiving red blood cells >10 U  from 2013 to 2016. The frequencies of platelet infusion were of 14.3% in 2013 to 66.7% in 2016 (P = .01), using tranexamix acid (trans-4-amino-methyl-cyclohexane-carboxylic acid) of 0% in 2013 to 86.7% in 2016 (P < .001) and postoperative hemoglobin levels of 87.0 ± 14.9 g/L in 2013 to 103.4 ± 20.1 g/L in 2016 (P = .003).Comparisons of blood components and outcome evaluation in patients with massive blood transfusion were shown in Table 3.5Logistic regression analysis suggests Table  that the4 In recent years, due to increasing use of second-line contraction drugs, uterine inertia induced PPH has been effectively controlled, while PPH caused by placenta previa and placental implantation has become more and more prominent.The purpose of intraoperative blood transfusion is to correct blood loss, supply erythrocytes, improve blood oxygen-carrying capacity, improve microcirculation, supplement blood colloidal components and blood coagulation factors, and maintain normal coagulation functions. Common causes for infusion of blood products during cesarean section are prenatal coagulation deficiency of blood components and PPH. The main causes of prenatal deficiency include prenatal anemia, preeclampsia, AFLP, and prenatal blood loss . PPH is a leading cause of maternal mortality, and is common in patients with postpartum uterine inertia and placental previa with or without placental implantation.This study showed that total volume of blood transfusion and number of blood transfusion increased year by year from 2013 to 2016, especially in cases of major bleeding (≥1000 ml) requiring massive blood transfusion (≥10 U). The study of Jie in China also showed that total transfusion volume, consumed erythrocyte, and plasma volumes increased significantly in 2015 and 2016 with the launching of the two-child policy. From our data analysis, causes of massive blood transfusions during cesarean section were 95.8% for placenta previa, and 93.1% for these who had history of cesarean section. Pernicious placenta previa refers to the occurrence of placenta previa with the placenta attached to the uterine scar. Its intraoperative bleeding is rapid and massive, and hemostasis is difficult, which usually requires massive blood transfusion, and even hysterectomy to control bleeding.,12,17,18 Furthermore, PPH associated with pernicious placenta previa is extremely dangerous, which would lead to major bleeding, hemorrhagic shock, DIC, and severe complications of the surrounding bladder, ureters, or intestines.,12 Cesarean section of pernicious placenta previa is a great challenge for obstetricians and anesthesiologist. Previous studies have shown that repeated cesarean section and placenta previa are independent risk factors for PPH, blood transfusion, and postpartum hysterectomy.–22 Our findings also suggest that in the context of the two-child policy, cesarean section rate in primiparas needs to be controlled, for reducing the occurrence of scarred uterus and blood transfusion associated with repeated cesarean section as well.While the trend of blood transfusion in the developed countries showed a decrease from 1996 to 2006. The traditional concept of blood transfusion is passive, conservative, and not timely, which is not suitable for patients with pernicious placenta previa who are expected to suffer from major bleeding.,12 In recent years, studies in China and elsewhere have shown that blood infusion at an early stage for patients with acute major bleeding can greatly improve success of rescue and reduce mortality, and the concept of massive transfusion protocol (MTP) was proposed.–27 It refers to the principle that blood transfusion is performed based on a pre-designed blood component protocol when major bleeding occurs, which requires active multidisciplinary collaboration of obstetrics, anesthesiology, and blood bank.In the traditional concept of intraoperative blood transfusion, volume expansion with crystalloids should be performed firstly for patients with bleeding. When bleeding is greater than 20% of total blood volume of patients, erythrocyte infusion should be considered, and when bleeding is greater than 30%, the infusion of blood components such as erythrocytes, plasma, and cryoprecipitate should be considered.9/L. Fortunately, there was no case of severe capillary hemorrhage and difficulty in hemostasis. The percentages of patients with postoperative coagulation abnormalities were 42.9%, 37.5%, 47.4%, and 40.0% in the year of 2013, 2014, 2015, and 2016, respectively. Although there were no serious complications such as DIC after cesarean section, the prolongation of PT and APTT, as well as the decrease of the fibrinogen were important issues that should be paid attention.In this study, there was no significant difference in the ratio of erythrocyte infusion and plasma infusion for patients with massive blood transfusion during 2013 to 2016. The percentage of intraoperative platelet infusion increased year by year. Changes in coagulation functions should receive more attention in patients with massive blood transfusion in addition to infusion of erythrocytes and plasma in proportion. For massive blood transfusion, coagulation factors were supplemented by infusion of cryoprecipitate. Reasonable infusion of platelets and cryoprecipitate is especially important to maintain the coagulation functions of the patients. Nevertheless, because there are few sources of platelets, clinical application is delayed and conservative. After cesarean section, there were still patients with platelets <50 × 10–30 An exploratory analysis of the CRASH-2 trial showed that the use of tranexamic acid at an early stage (<3 hours) can reduce the mortality due to bleeding in patients with trauma. Application of tranexamic acid in patients with PPH can also reduce the bleeding volume, potentially reducing blood transfusion and avoiding hysterectomy. On the other hand, the results of a World Maternal Antifibrinolytic trial showed that the use of tranexamic acid did not lead to an increase in thrombotic events, and the study recommended that it was reasonable to use tranexamic acid during severe PPH or when severe bleeding were expected to occur. At our center, tranexamic acid is generally administered intravenously at 1.0 g when bleeding ≥1000 ml based on the recommendations of previous references and guidelines,–36 and there is risk of continuous bleeding, but no consensus in clinical practice for the exact use of tranexamic acid, which should be further investigated in clinical experiments. In this study, no patient suffered from thrombotic event after the use of tranexamic acid.The importance of antifibrinolytic drugs is gradually recognized in improving coagulation functions in patients with major bleeding. In this study, there was only 1 case used tranexamic acid in 2014, while the tranexamic acid use rate in 2015 and 2016 increased significantly compared to the years of 2013 and 2014. Tranexamic acid is a synthetic lysine analog that binds to plasminogen in order to block its activation into plasmin. Tranexamic acid was developed in 1962 in the course of seeking medical treatment for PPH.In this study, the difference in hemoglobin before and after cesarean section was statistically significant only in 2014. However, the average values of hemoglobin after the operation in 2015 and 2016 were slightly higher than those before the operation. Moreover, longitudinal comparison showed that hemoglobin in 2015 and 2016 was significantly higher compared to 2013. At first, blood transfusion was passive and the amount of blood transfusion was conservative to correct blood loss in patients with pernicious placenta previa due to limited awareness of the risk. After recognizing the risk of major bleeding to pernicious placenta previa, passive response was replaced by active response to major bleeding since the end of 2014. Therefore, postoperative hemoglobin can be maintained at a higher level.The limitations of this study are that the patients were all from a single hospital, limiting the generalizability of the results. The 4 years is a relatively short period and the investigation time needs to be extended. Finally, this study is retrospective and therefore cannot analyze unrecorded factors in medical charts. In the future, we will focus on the cooperation of MDT to predict preoperative blood loss and strategies to reduce blood loss. The most important method is to reduce the rate of primary cesarean delivery to reduce the occurrence of morbidly adhesion of the placenta.5The two-child policy in China led to an increase popularity of repeat cesarean section. The frequency of placenta previa accompanied with previous cesarean section increased the need for massive blood transfusion. Our results indicate that the indications of cesarean section should be strictly limited to avoid unnecessary cesarean section for primipara. Repeat cesarean section is usually used for previous cesarean section. Therefore, it effectively reduces the dangerous placenta previa caused by uterine scars in limiting primary cesarean section, thereby reducing the rate of massive blood transfusion.Conceptualization: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Data curation: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Formal analysis: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Funding acquisition: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Investigation: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Methodology: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Project administration: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Resources: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Software: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Supervision: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Validation: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Visualization: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Writing – original draft: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.Writing – review & editing: Changqing Zhou, Li Zhang, Yang Bao, Ling Li, Ting Zhang, Xiyan Zhang, Chunling Wang.


Prostate cancer is a common malignancy in men worldwide. Lysophosphatidic acid receptor 1 (LPAR1) is a critical gene and it mediates diverse biologic functions in tumor. However, the correlation between LPAR1 and prognosis in prostate cancer, as well as the potential mechanism, remains unclear. In the present study, LPAR1 expression analysis was based on The Cancer Genome Atlas (TCGA) and the Oncomine database. The correlation of LPAR1 on prognosis was also analyzed based on R studio. The association between LPAR1 and tumor-infiltrating immune cells were evaluated in the Tumor Immune Estimation Resource site, ssGSEA, and MCPcounter packages in R studio. Gene Set Enrichment Analysis and Gene Ontology analysis were used to analyze the function of LPAR1. TCGA datasets and the Oncomine database revealed that LPAR1 was significantly downregulated in prostate cancer. High LPAR1 expression was correlated with favorable overall survival. LPAR1 was involved in the activation, proliferation, differentiation, and migration of immune cells, and its expression was positively correlated with immune infiltrates, including CD4+ T cells, B cells, CD8+ T cells, neutrophils, macrophages, dendritic cells, and natural killer cells. Moreover, LPAR1 expression was positively correlated with those chemokine/chemokine receptors, indicating that LPAR1 may regulate the migration of immune cells. In summary, LPAR1 is a potential prognostic biomarker and plays an important part in immune infiltrates in prostate cancer. Prostate cancer is a common malignancy in men worldwide . It is aLysophosphatidic acid receptor 1 (LPAR1) is one of the G protein-coupled receptors and binds with lysophosphatidic acid (LPA) . It is iThe rapid development of high-throughput sequencing makes it possible to explore the mechanisms in diseases . The CanIntegrative analysis and several visualization methods were used in this present study to explore the mechanism of LPAR1 in prostate cancer. We investigated the LPAR1 expression levels and analyzed the correlation of LPAR1 and the prognosis of patients. Gene Ontology (GO) analysis, Gene Set Enrichment Analysis (GSEA), and several methods were also utilized to explore the potential function of LPAR1 in tumor progression and immune microenvironment. The findings suggested the potential mechanisms of LPAR1, giving us new insights into the important role of LPAR1 in prostate cancer.https://tcga-data.nci.nih.gov/tcga/) through the TCGAbiolinks (The prostate adenocarcinoma (PRAD) clinical and molecular data (including mRNA expression and mutations) was extracted from the TCGA Data Portal (biolinks  R packaghttps://www.oncomine.org/). We screened the mRNA levels of LPAR1 in various types of cancers. P-value <0.05 and fold change >2 were restricted as the thresholds.The Oncomine microarray database was used for analysis . The TIMER includes more than 10,000 samples across multiple cancer types of the TCGA. It applies a partial deconvolution linear least square regression method to calculate the abundance of immune infiltrates. We evaluated the correlation between LPAR1 expression and immune infiltrates in tumors, including CD8+ T cells, CD4+ T cells, dendritic cells, neutrophils, B cells, and macrophages.The Tumor Immune Estimation Resource (TIMER)  is an inThe Microenvironment Cell Populations counter (MCPcounter) method  and singvia the STRING database (https://string-db.org/). The minimum required interaction cutoff is 0.4. The edges between nodes represent protein–protein associations. The edge with blue color means that the two nodes have known interactions from curated databases. The yellow color means textmining. The black color means that the two nodes have a co-expression. The purple color means that the interactions of the two nodes were experimentally determined.The LPAR1 and chemokines/chemokine receptors were searched in a protein–protein interaction (PPI) network P < 0.05.The statistical analysis and the graphical work in this study were mainly conducted by R programming language with several packages, such as DEseq2 package, survival package, and TCGAbiolinks package. The survival curve based on log-rank test was depicted with Kaplan–Meier method. Univariate survival analysis was based on Cox proportional hazards model. Hazard ratio (HR) and log-rank test were used for comparing the overall survival between patients in different groups. Throughout the study, the threshold of statistical significance was via TIMER database and show it in To compare the mRNA expression levels of LPAR1 in normal and tumor tissues, we used the Oncomine database to determine the LPAR1 expression among multiple cancer types. This analysis indicated that LPAR1 was highly expressed in lymphoma and lowly expressed in prostate cancer, bladder cancer, brain and central nervous system cancer, head and neck cancer, colorectal cancer, kidney cancer, lung cancer, leukemia, melanoma, and ovarian cancer . To furtr = −0.475, P = 7.54e−25), Furthermore, the LPAR1 expression was positively correlated with the abundance of several immune cell types, including CD4+ T cells , CD8+ T cells , neutrophils , macrophages , and dendritic cells  in PRAD. To validate these findings, we used the MCPcounter method. We evaluated the association between LPAR1 and tumor-infiltrating immune cells from the mRNA expression data. A strong positive correlation between LPAR1 and myeloid dendritic cells, T cells, B lineage, monocytic lineage, cytotoxic lymphocytes, and natural killer (NK) cells was seen  in prostate cancer, suggesting that LPAR1 expression can impact the prognosis of patients with prostate cancer.The downregulation of LPAR1 was validated by using the TCGA-PRAD dataset . To gainPatients with high LPAR1 expression had a prolonged OS time, suggesting that LPAR1 may be involved in the initiation and the progression of prostate cancer. Then, we analyzed the RNA sequencing data downloaded from TCGA and compared the tumor samples between samples with high LPAR1 expression and samples with low LPAR1 expression. The volcano plot was shown in Furthermore, we found that LPAR1 can take part in several immune-related pathways and influence many processes of immune cells. The results indicated that LPAR1 was associated with the activation  of T celTo further clarify the association between LPAR1 and immune cell migration, we integrated chemokines and chemokine receptors in To better understand the interactions among LPAR1 and chemokines or chemokine receptors, the STRING database was utilized to generate and visualize a PPI network. The PPI network showed that LPAR1 had known or predicted interactions with the various chemokines studied above , includiThe present study suggests, based on bioinformatics analysis, the importance of LPAR1 in prostate cancer. LPAR1 was lowly expressed in prostate cancer and was significantly related to patient survival. In addition, LPAR1 may be involved in the biological process when immune cells move into the tumor tissues and improve the TME of patients, which impact the development of prostate cancer and the prognosis of patients. Hence, LPAR1 is a potential immune-related biomarker in prostate cancer. Our findings offer deeper insights into the mechanisms of LPAR1 in the development of prostate cancer.In this study, based on independent datasets in the Oncomine database and TCGA datasets, we examined the LPAR1 expression level in various types of cancer. The differential expression of LPAR1 was seen in a set of cancers between tumor and normal tissues. In the Oncomine database, the results showed that LPAR1 was highly expressed in lymphoma while lowly expressed in prostate, bladder, brain, colon, head and neck, kidney, leukemia, lung, melanoma, and ovarian cancers. The TCGA datasets showed that LPAR1 expression was significantly lowly expressed in PRAD, BLCA, BRCA, COAD, READ, ESCA, HNSC, KICH, KIRC, LIHC, STAD, THCA, and UCEC compared with normal tissues.The TME has a great impact on the development of cancers –6, 31. SConcerning biological function, LPAR1 participated in many signaling pathways in tumor cells through GO analysis, for example, passive transmembrane transporter activity, cell motility, metal ion transport, cell differentiation, tube development, G protein-coupled receptor signaling pathway, and chemotaxis. The GSEA results showed that the LPAR1 function was enriched in GO_Leukocyte_Differentiation and GO_Lymphocyte_Mediated_Immunity in prostate cancer. Furthermore, the GSEA on LPAR1 immune-related function indicates that LPAR1 influenced the activation, proliferation, differentiation, and migration of immune cells. It hints that LPAR1 improves the immune response of prostate cancer through various pathways.LPAR1 has been proven to be associated with chemotaxis, which was also found in the present study. Studies had reported that LPAR1 played a critical role in the LPA-induced chemotactic migration of olfactory ensheathing cells . LPAR1-dAs far as we know, this is the first study to elaborate the potential functions of LPAR1 and its association with tumor-infiltrating immune cells by using integrated bioinformatics analysis. However, this present study had limitations. Further molecular experiments are deserved to verify the mechanisms of LPAR1 and its effects on the clinical outcome in prostate cancer. Moreover, it is also important to integrate and elaborate the association between LPAR1 and chemokines/chemokine receptors, which can help us better understand the TME, especially the immune microenvironment in tumors.https://tcga-data.nci.nih.gov/tcga/) and Oncomine (https://www.oncomine.org/) websites, so the ethical statement was already admitted and further evaluation of it was not necessary.All datasets were extracted from The Cancer Genome Atlas (KY and JS conceived and designed the project. JS, DJ, SY, XZ, JW, YLi, YS, and YLu analyzed the data. JS wrote the manuscript with input from all the other authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Herpes zoster (HZ), is a painful skin rash disease with cutaneous symptoms and acute zoster-associated pain (ZAP). Postherpetic neuralgia (PHN), as the most frequent sequela of HZ, can persist a long time. Both HZ and PHN may significantly impact the quality of life and made great economical afford to affected patients. Its optimal treatment on HZ and PHN is still an urgent problem. In China, thermotherapy, including moxibustion and fire needle, is widely used because they can quickly promote the recovery of shingles and reduce the occurrence of PHN. Thermotherapy can also reduce pain intensity, relieve anxiety, and improve quality of life of PHN. Based on the current literatures, the effect and safety of thermotherapy will be systematically evaluated to provide appropriate complementary therapies for HZ and PHN.I2 > 50%).Studies search for eligible randomized controlled trials (RCTs) that use thermotherapy including fire needle and moxibustion for HZ or PHN from the following databases: PubMed, EMBASE, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), China Biology Medicine Database (CBM), Technology Periodical database (VIP), and Wanfang database. Language restrictions for retrieving literature are English and Chinese. Their data extraction will be done by 2 researchers. Mean difference (MD) or relative risk (RR) with fixed or random effect model in terms of 95% confidence interval (CI) will be adopted for the data synthesis. To evaluate the risk of bias, the Cochrane's risk of bias assessment tool will be utilized. The sensitivity or subgroup analysis will also be conducted when meeting high heterogeneity (This meta-analysis will provide an authentic synthesis of the thermotherapy's effect on HZ and PHN, including incidence of postherpetic neuralgia and adverse events.The findings of the review offer updated evidence and identify whether thermotherapy can be an effective treatment for HZ and PHN for clinicians.INPLASY2020110009. Cutaneous symptoms and acute zoster-associated pain (ZAP) affects quality of life (QoL) of the affected patients. It is found that the pain and discomfort associated with HZ and PHN can have a significantly negative and widespread impact on patients’ health related quality of life (HRQoL) and patients’ ability to engage in activities of daily living.–5 Therefore, HZ and PHN made great economic burden. The healthcare costs and loss of production costs were between 100 and 175 euros per 3 months for the significant pain group after HZ in Netherlands. Some patients with PHN last for more than 10 years, affecting sleep and causing depression. Exclude immunocompromised patients, 6.6% of patients ≥50 years with HZ developed PHN in USA. PHN patients range from 50s to 80s spend from $6133 to $11,147 every year, which increases with age. 30% to 50% of patients with PHN experienced pain lasting for more than 1 year. It is necessary to find effective, low-toxic, and easy to operate treatments.Postherpetic neuralgia (PHN) is refractory neuralgia that can persist for a long time and has severe pain left after the onset of HZ. A systematic review concealed that the incidence rate of HZ ranged between 3 and 5/1000 person-years in North America, Europe, and Asia-Pacific.–10 The use of antiviral medications and glucocorticoids does not reduce the incidence of PHN. Thermotherapy, including fire acupuncture and moxibustion, have been used in clinical practice for more than 2000 years, recorded in Huangdi Nei Jing. Records of herpes zoster symptoms in ancient Chinese books appeared in the Pre-qin Dynasty's “Fifty-two prescriptions” earlier than Huangdi Nei Jing. Acupuncture was recommended as the treatment method for HZ and PHN in the 2018 Chinese Consensus of Herpes Zoster and the 2016 Chinese Consensus of Diagnosis and Treatment of Postherpetic Neuralgia.,14 Acupuncture may reduce pain intensity, relieve anxiety, and improve quality of life in patients with PHN. Several researches proved fire acupuncture or moxibustion can significantly relieve pain, shorten the pain duration, and reduce the incidence of postherpetic neuralgia.,17However, there was no obvious effective Western medicine treatment on HZ and PHN and Western medicine has certain side effects.However, there are few meta-analysis on the effect of fire acupuncture and moxibustion on HZ or PHN. The purpose of this meta-analysis is to assess the quality of current randomized controlled trials and further systematically evaluate the effectiveness and safety of fire acupuncture and moxibustion. This research may provide useful information in forming an optimal fire acupuncture or moxibustion treatment protocol.2https://doi.org/10.37766/inplasy2020.11.0009). The protocol follows the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) statement guidelines. We will describe the changes in the full review if necessary.The protocol for this systematic review was registered on INPLASY (INPLASY2020110009) and is available in full on the inplasy.com  of thermotherapy including fire needle and moxibustion as the treatment in English and Chinese for Herpes zoster or PHN will be involved in the review.2.1.2Patients included will be clinically diagnosed with herpes zoster or PHN and there is no restriction on sex, age, race, or duration of the disease. Besides, studies that do not mention diagnostic criteria and with other specific primary diseases will be excluded.2.1.3One of thermotherapy, fire acupuncture, and moxibustion must be applied in the treatment group, while Western medicine must be applied in the control group with all types of point combination being acceptable.2.1.42.1.4.11.Total effective rate;2.Pain intensity ;3.Recovery span of skin damage;4.The duration of the pain.2.1.4.21.Incidence of postherpetic neuralgia;2.Adverse events.2.22.2.1The data collection will be conducted systematically by 2 researchers through 8 databases from their inception to Feb 5, 2020. The databases to be searched for eligible RCTs are as follows: PubMed, EMBASE, Web of Science, the Cochrane Library, China National Knowledge Infrastructure (CNKI), China Biology Medicine Database (CBM), Technology Periodical database (VIP), and Wanfang database. A combination of keywords, written in English and Chinese, will be searched, including: “thermotherapy,” “fire needle,” “moxibustion,” “herpes zoster,” and “postherpetic neuralgia.”2.2.2Despite published studies in journals, literatures from conference papers and references, will be searched.2.32.3.1The inclusion and exclusion of studies will be accomplished by 2 reviewers (ZkL and ZL) independently and be crosschecked. First, reviewers will review the titles and abstracts of the literatures and exclude the markedly ineligible ones. Second, the full text will be carefully inspected for inclusion in the RCT trials through inclusion and exclusion studies. Any disagreement will be resolved through discussion with the third researcher. The whole selection process will be presented in a PRISMA flow diagram Fig. .2.3.2Study characteristics, details of the intervention and comparator used, outcome measures, results, and risk of bias ratings will be extracted by 2 individuals working independently, stored in Excel files and analyzed using RevMan 5.3 and Stata 13.0. Inconsistencies will be resolved through discussion and consultation with a third researcher.2.3.3When meeting problematic data which is ambiguous, contradictory, incorrect, or missing, the researcher will contact the first author for the clarified, correct, or omission data. The potential impact of suspicious data will be discussed additionally in Section 3.2.3.4Two reviewers will independently evaluate the methodological quality of included studies using the Cochrane Handbook's Risk of Bias Tool. Seven sectors will be evaluated, including random sequence generation, allocation concealment, blind participants and personnel, blind evaluation of results, completeness of result data, selective reporting, and other sources of bias. The reviewers will use 3 letters to indicate the results of the 3 levels of evaluation: “L” (low-risk), “U” (uncertain), “H” (high-risk). When there is different idea between 2 reviewers, a discussion with a third reviewer will be needed and the corresponding author will be connected when needed.2.3.5Evaluating the effect of continuous data, the mean difference (MD) expressed as a 95% confidence interval (CI) will be used, just like the relative risk (RR) of dichotomous data.2.3.6I2 statistics, a random effect model or a fixed effect model is adopted. Specifically, if the heterogeneity is high (I2 > 50%), a fixed effect model will be used. Otherwise, a random effects model will be applied.According to the data heterogeneity analysis, given by the 2.3.7P > .05 will be interpreted as no substantial reporting bias. Since the asymmetry of the funnel chart cannot replace publication bias, the potential reasons will be distinguished by small sample size, low methodological quality, or true heterogeneity.When the study is sufficient (at least 10 RCTs), the funnel graph will visually show the reported bias. If it shows asymmetry, then the test of Begg and Egger will be completed, and the value of 2.3.8The systematic review will be conducted with the use of RevMan 5.3. Taking account of the heterogeneity assessment, MD or RR with fixed or random effect model will be computed. Additionally, if heterogeneity is considered significant, the sensitivity or subgroup analysis will be generated to distinguish the source of it. When it comes to the situation that the data are insufficient for quantitative analysis, the review will only represent and summarize the evidence.2.3.9As mentioned above, sensitivity analysis will be performed when the heterogeneity is greater than 50%. Specifically, despite studies of methodological quality, sample size, or older age, effect of missing data, a meta-analysis will be conducted again to determine whether these factors will affect the results.2.3.10I2 > 50%), a subgroup analysis will also be performed. Participant characteristics such as age and gender, pain degree, study quality, ethnicity, outcome measures, and other characteristics will become the content of the subgroup analysis.After observing the obvious heterogeneity ” and fill out the “Summary of Survey Results” form.2.3.12Since this protocol is for a systematic review, which involves no privacy data, ethical approval, and informed consent are needless. The results of the review will be widely disseminated through publications and conference presentations submitted to peer review.3r = 0.846), sleep (r = 0.774), and QoL.,18Shingles (herpes zoster) is an infectious skin disease caused by the reactivation of varicella-zoster virus (VZV), which is latent in the posterior root ganglion or cranial ganglion of the spinal cord. Researches proved that the degree of pain is closely related to mood disturbance (–7,19 and there is remaining considerable confusion about the optimal treatment for HZ and PHN.The treatment goal of herpes zoster is to relieve pain in the acute phase, shorten the duration of the lesion, prevent the lesion from spreading, and prevent or reduce PHN and other complications. The purpose of PHN treatment is to effectively control pain as early as possible, relieve accompanying sleep and affective disorders, and improve the quality of life. However, their efficacy is still limited by Western medicine and have several adverse events,20,21 Fire needle could increase level of adenosine and AMP and ADP to release pain. The internal mechanism to treat the PHN patients is to possibly change the protomics indexes like MFG-ES, Lymphotoxin beta/TNFSF3, IL-19, Neuritin, NCAM-1/CD56, and PECAM-1/CD31 by the Zhuang medicine medicated thread moxibustion.In recent years, clinical practices and some studies have also found that thermotherapy, including fire acupuncture and moxibustion could offer great benefits to HZ and PHN and thermotherapy is safe and have few adverse events.However, there is no available systematic review for the effectiveness and safety of thermotherapy in patients with HZ or PHN. We hope this systematic review can help comprehensively to evaluate its efficacy and safety, and contribute to the management and mechanism of HZ or PHN.Guohua Lin is the guarantor of the article and will be the arbitrator when meeting disagreements. All research members participated in developing the criteria and drafting the protocol for this systematic review. YS, JZ, ZkL, and ZL established the search strategy. YS, ZL will independently accomplish the study selection, data extraction, and assess the risk of bias. ZkL, JZ will perform the data syntheses. The subsequent and final versions of the protocol are critically reviewed, modified, and authorized by all authors.Conceptualization: Guohua Lin.Data curation: Zhenke Luo, Jingchun Zeng.Formal analysis: Zhuang Li, Yalin She.Funding acquisition: Guohua Lin, Jingchun Zeng.Methodology: Guohua Lin, Jingchun Zeng.Project administration: Guohua Lin.Software: Yalin She, Zhenke Luo.Supervision: Wenya PEI, Jingchun Zeng.Validation: Zijun Liu.Visualization: Wenya PEI, Zhuang Li.Writing – original draft: Zhuang Li, Yalin She, Zhenke Luo, Zijun Liu.Writing – review & editing: Guohua Lin, Jingchun Zeng.


Idiopathic hypoparathyroidism (IHP) is a rare disorder that is diagnosed by excluding other possible etiologies. Thyroid surgery causes approximately 14–60% of all cases of hypoparathyroidism; of these, surgery for papillary thyroid carcinoma (PTC) is the most common reason. Here, we report an extremely rare case of IHP combined with PTC.A 22-year-old man presented with a history of uncontrollable extremity and facial numbness, spasm and twitch lasting for nine years. He had been misdiagnosed with epilepsy and gained no relief from antiepileptic therapy. The laboratory evaluation revealed reduced parathyroid hormone and serum calcium and elevated inorganic phosphorus. After considering IHP, ultrasound detected a solid hypoechoic and irregularly shaped nodule 13×8×9 mm in size in the upper pole of the right thyroid gland, and fine-needle aspiration biopsy indicated PTC. Then, the patient underwent surgical treatment and radioactive iodine ablation. The long-term treatment strategy consisted of oral levothyroxine for thyroid-stimulating hormone inhibition and oral calcium and vitamin D supplements for hypocalcemia control.We report a rare case of IHP combined with PTC in a 22-year-old male. Some experiences and lessons from our treatment procedure merit discussion, and we hope that our report can serve as a reference for the diagnosis and treatment of similar patients in the future. Hypoparathyroidism is an uncommon endocrine disorder characterized by the absence or biological inactivity of parathyroid hormone (PTH), followed by hypocalcemia, hyperphosphatasemia and increased urinary calcium excretion , 2. The Thyroid carcinoma, especially papillary thyroid carcinoma (PTC), is the most common endocrine malignancy, and its incidence has dramatically increased in recent decades . While sIn February 2019, a 22-year-old man presented to our emergency department with uncontrollable extremity and facial numbness, spasm and twitching lasting for half an hour. He had a history of the above-mentioned symptoms for 9 years and aggravation for 1 year . Previously, multiple electroencephalogram examinations were performed at his local hospital, and the results showed occasional short-term sharp waves in his forehead. In the absence of laboratory tests of parathyroid hormone (PTH) and serum calcium, he was empirically diagnosed with epilepsy and treated with oral sodium valproate  without improvement; hence, he decided to stop taking the medicine 6 months prior.Candida and Pneumocystis carinii, and an absolute count of peripheral blood lymphocytes in the normal range; thus, the possibility of congenital hypo-parathyroidism, such as De George’s syndrome, was excluded. Furthermore, no abnormal family history or autoimmune history was presented. Given the patient’s obvious extremity and facial numbness and spasm and because the laboratory tests showed hypoparathyroidism, hypocalcemia and hyperphosphatasemia, after excluding other possible etiologies, a diagnosis of IHP was suspected, and his symptoms were alleviated by urgent intravenous calcium gluconate (20 mg IV).After visiting our hospital, the laboratory examination upon admission revealed the following: PTH 1.34 pmol/L , serum calcium 1.55 mmol/L , inorganic phosphorus (IPOS) 1.84 mmol/L , magnesium 0.84 mmol/L  and 25-hydroxyvitamin D3 63.11 nmol/L . The urinary system ultrasonography was negative. He had no history of neck surgery, neck radiotherapy, parathyroid disease or hereditary disease. In addition, the patient had no special facial features, no history of macrovascular abnormalities, no history of serious fungal infection, such as To comprehensively evaluate the condition of IHP, cervical ultrasonography was performed and showed negative results for the parathyroid glands, but a solid hypoechoic mass measuring 13×8×9 mm with irregular margins, macrocalcifications, a taller than wide shape and suspicious extracapsular extension was accidentally found in the upper pole of the right thyroid gland Figure 1th ATA guidelines, for patients with PTC >1 cm and <4 cm without high invasive characteristics, the initial surgical procedure can be either total thyroidectomy or lobectomy. To reduce the risk of postoperative hypoparathyroidism, hemithyroidectomy was determined as a basic surgical procedure. However, the primary premise is a sufficient surgical resection range to avoid the recurrence of the tumor and a secondary operation, which may also increase the risk of parathyroid injury. Therefore, the final resection range should be comprehensively judged by the preoperative and intraoperative findings. Besides, the protection of parathyroid gland during operation lies in careful anatomy and avoidance of parathyroid and supplied vascular injury.Multidisciplinary discussions focused on how to address PTC and protect the parathyroid function in the presence of IHP. Given that the size of the primary lesion was greater than 1 cm, active surveillance and thermal ablation were ruled out, and surgical intervention was finally considered. According to the 2015As mentioned above, sufficient preoperative evaluations, including neck ultrasonography, thyroid function tests and FNA, were performed, and normal bilateral vocal cord activity was confirmed by preoperative vocal cord laryngoscopy. In addition, the serum calcium and PTH levels were evaluated again, and the PTH value was still at an abnormally low level (1.26 pmol/L), while the serum calcium value returned to within the normal range (2.24 mmol/L) after the calcium and calcitriol supplementation.During the operation, first, right hemithyroidectomy and isthmusectomy were performed. After dissecting the isolated thyroid gland evenly according to a thickness of 1-2 mm, a hard, white nodule measuring approximately 13×10×10 mm in size was found in the upper pole of right thyroid gland. Moreover, several fused LNs ranging in size from 2 to 12 mm were found in the right central and pretracheal areas with a maximum diameter of 12×10×8 mm. Then, the tissues in the right central and pretracheal areas were examined by intraoperative cryosectioning, and a high LN metastasis ratio was found . Considering the possibility of LN metastasis in the contralateral central area, the patient could have needed RAI ablation after the operation; thus, total thyroidectomy plus bilateral CND were finally performed. After the excision of the tissue, a careful search in the dissected thyroid (slice thickness 1–2 mm) and central tissue was routinely performed to identify mistakenly cut parathyroid glands. During the operation, only one suspicious parathyroid was found in the superior left position Figure 2The postoperative paraffin section examination showed a hard, grayish-white nodule measuring 13 mm in diameter with an extracapsular extension in the right thyroid gland, and the histological findings showed that the tumor cells were arranged in branched papillary structures, and the nucleus was ground glass-like with pseudoinclusion bodies and nuclear grooves; granular calcification was scattered between the tumor cells. Furthermore, a high metastatic ratio was found in the central LNs , which ranged in diameter from 2–12 mm. A protocol of oral levothyroxine 100 µg qd was applied for long-term TSH inhibition. The postoperative calcium supplementation strategy consisted of calcium gluconate 20 mg IV bid and oral calcitriol 0.25 µg bid until discharge. Subsequently, the protocol was changed back to oral Caltrate D 600 mg bid and oral calcitriol 0.25 µg qd for the long term.131 whole-body diagnostic imaging with a dose of 5 mCi revealed no abnormal radiation uptake anywhere in the body, except for the thyroid bed area; thus, RAI therapy was considered. Subsequently, RAI therapy with 150 mCi was performed. The patient was discharged 4 days after the treatment, and despite the long-term follow-up, the hTg value remained outside of the desired range recommended by the 2015th ATA guidelines  21.23 µg/L, and idelines Figure 3During the follow-up period, the serum calcium and PTH values were continuously monitored, and the variation curve is shown in To the best of our knowledge, this is the first report of a patient with IHP combined with PTC, and we describe the treatment procedure and initial outcomes in great detail. In this rare case, the patient was initially empirically misdiagnosed with epilepsy at a local medical unit and received antiepileptic treatment for up to nine years. After the diagnosis of IHP and PTC, the patient underwent surgery and RAI for PTC, followed by long-term TSH inhibition therapy and oral calcium and vitamin D supplementation.This case underscores several critical points that should be considered. First, hypoparathyroidism and hypocalcemia should be considered in patients with unexplained convulsive symptoms, and routine examinations of calcium, phosphorus and PTH are economical and effective for diagnosis and differential diagnosis, especially when other treatments fail to achieve the desired goals. In addition, extensive central nervous system calcification is common in patients with hypoparathyroidism; these lesions can be detected by CT, and prevalence rates of 52–74% have been previously reported , 14. IHPSecond, imaging evaluations should not be ignored when evaluating functional organs. Generally, the diagnosis of hypoparathyroidism depends on low PTH levels in the context of hypocalcemia, and imaging evaluations are not routinely suggested in the guidelines , 16. Howth ATA guidelines, active surveillance is suggested only for select low-risk papillary thyroid microcarcinoma (PTMC) patients , Post-Doctor Research Project, West China Hospital, Sichuan University (2019HXBH043), and Sichuan Province Science and Technology Project of China (2020YFS0208).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.


Background: Atherosclerosis manifests as a focal disease, often affecting areas with complex hemodynamics such as the carotid bifurcation. The magnitude and regularity of the hemodynamic shear stresses acting on the vessel wall are thought to generate risk patterns unique to each patient and play a role in the pathogenesis of atherosclerosis. The involvement of different expressions of shear stress in the pathogenesis of carotid atherosclerosis highlights the need to characterize and compare the differential impact of the various expressions of shear stress in the atherosclerotic carotid bifurcation. Therefore, the aim of this study is to characterize and compare hemodynamic wall shear stresses (WSS) in the carotid arteries of subjects with asymptomatic atherosclerotic plaques. Shear stresses were also compared against vessel diameter and bifurcation angle to examine the relationships with the geometry of the carotid bifurcation.Methods: 4D Flow MRI and contrast-enhanced MRA data were acquired for 245 subjects with atherosclerotic plaques of at least 2.7 mm in conjunction with the Swedish CArdioPulmonary bioImage Study (SCAPIS). Following automatic segmentation and geometric analysis, time-resolved WSS and near-wall turbulent kinetic energy (nwTKE) were derived from the 4D Flow data. Whole-cycle parameters including time-averaged WSS and nwTKE, and the oscillatory shear index (OSI) were calculated. Pairwise Spearman rank-correlation analyses were used to investigate relationships among the hemodynamic as well as geometric parameters.Results: One hundred and seventy nine subjects were successfully segmented using automated tools and subsequently geometric and hemodynamic analyses were performed. Temporally resolved WSS and nwTKE were strongly correlated, ρ = 0.64. Cycle-averaged WSS and nwTKE were moderately correlated, ρ = 0.57. Cycle-average nwTKE was weakly correlated to OSI (ρ = −0.273), revealing that nwTKE provides information about disturbed flow on the vessel wall that OSI does not. In this cohort, there was large inter-individual variation for both WSS and nwTKE. Both WSS and nwTKE varied most within the external carotid artery. WSS, nwTKE, and OSI were weakly correlated to vessel diameter and bifurcation angle.Conclusion: The turbulent and mean component of WSS were examined together in vivo for the first time, and a strong correlation was found between them. nwTKE presents the opportunity to quantify turbulent wall stresses in vivo and gain insight into the effects of disturbed flow on the vessel wall. Neither vessel diameter nor bifurcation angle were found to be strongly correlated to the turbulent or mean component of WSS in this cohort. Atherosclerosis manifests as a focal disease, predominantly affecting areas with complex hemodynamics such as the outer walls of vessel bifurcations, recirculation zones, and areas of stasis –3. The cin-vitro, and may trigger plaque rupture through erosion of the plaque's fibrous cap  –20. Invein vivo data available to examine the relationship between disturbed flow and vessel geometry. In particular, the vessel diameter and the bifurcation angle of the carotid arteries are pointed to as potential surrogate markers of disturbed flow. Therefore, the secondary aim of this study was to examine the relationships between WSS, OSI, and nwTKE and the geometry of the carotid bifurcation and determine if these geometric factors can be used for prediction.The geometric risk hypothesis for atherosclerosis assumes a direct relationship between exposure to disturbed flow and the geometry of the vessel , 32. UnfSubjects were recruited as part of the Swedish CArdioPulmonary bioImage Study (SCAPIS) . Two hunMRI data was acquired with a 3T Philips Ingenia scanner  using an 8-channel dedicated carotid coil .3 and matrix size 512 × 512 × 100 set to cover the carotid arteries from the clavicle to the circle of Willis, flip angle 27°, echo time 1.8 ms, repetition time 4.9 ms, parallel imaging (SENSE) factor 2, and reconstructed spatial resolution of 0.48 × 0.48 × 0.50 mm3.Contrast-enhanced MR angiography (CE-MRA) data was acquired post injection of a gadolinium-based contrast agent  to generate bright-blood images for automated segmentations of the vessel lumen and basic geometric analyses. Scan parameters included: a coronal slab with 3D field of view = 200 × 200 × 50 mm3 and matrix size 192 × 192 × 19 set to cover the carotid arteries from the clavicle to the Circle of Willis, resulting in an acquired spatial resolution of 1.1 × 1.1 × 1.2 mm3. The data was acquired with an approximately isotropic spatial resolution was used to avoid directionally dependent results, as WSS and nwTKE measurements are dependent on spatial resolution. Additional parameters included: flip angle 10°, echo time 3.5 ms, and velocity encoding range (VENC) 1.2 m/s. VENC was chosen to avoid velocity aliasing and maintain optimum sensitivity for the measurement of TKE. SENSE parallel imaging was used with in-plane and out-of-plane acceleration factors of 1.6. Repetition time was 6.1 ms and two k-space lines were acquired per cardiac cycle, resulting in an acquired temporal resolution of 49 ms. Data was reconstructed to 40 timeframes using a temporal sliding window approach. Total scan time was 8 min. Data was corrected for concomitant gradient field effects on the scanner, while phase-wrapping and background phase-offset errors were corrected offline. Background phase-offset errors were corrected using a weighted 4th order fit to static tissue , external carotid artery (ECA), and internal carotid artery (ICA) using a 3D convolutional neural network (CNN), built upon on the DeepMedic framework , 37. MasThe automatically generated segmentations of the CCA, ECA, and ICA were additionally used to extract geometric descriptions of the carotid bifurcation. For each bifurcation, the mean diameter of each branch and the bifurcation angle were calculated. The bifurcation angle was calculated using vectors aligned with the centrelines of the ICA and ECA. Both vectors originated at the centreline point nearest to the bifurcation on each branch and intersected the midpoint of the centreline for each branch. Geometric analysis was performed using in-house software developed for MATLAB.4D Flow data was spatially up-sampled using cubic interpolation and subsequently registered to the CE-MRA images to facilitate analysis using the segmentations generated by the CNN. Registration was performed using the Elastix registration toolbox , 39. RegWSS was calculated using a previously described method , 22, 24.tWSS was investigated based on measurements of near-wall TKE (nwTKE). nwTKE was calculated using a previously described method . In shorWSS and nwTKE were calculated throughout the cardiac cycle. Descriptive statistics including the minimum, median, mean, standard deviation, and maximum values of WSS and nwTKE were extracted throughout the cardiac cycle, for each carotid branch. The same statistics were calculated for the time-averaged WSS (TAWSS) and time-averaged nwTKE (TAnwTKE), for each branch. The minimum and maximum values of WSS and nwTKE were computed as the 10% and 90% values, respectively, in order to reduce the impact of noise-related outliers. In addition, the oscillatory shear index (OSI) was calculated, for each branch . The TAW3, and dynamic viscosity was estimated as 3.2 × 10−3 pa·s.The Reynolds number was also calculated for each vessel, through time, using the estimated vessel diameter and vessel mean velocity at that timeframe. Blood was estimated to have a density of 1.060 kg/mt-tests were used to determine whether or not there were differences between hemodynamic parameters per branch using a significance level (α) of 0.05.Pairwise analyses using the Spearman rank correlation coefficient, ρ, were used to examine the relationships between WSS, OSI, and nwTKE parameters. Pairwise analyses using the Spearman rank correlation coefficient were also used to examine the relationships between geometric descriptions of the carotid bifurcation and hemodynamic stress parameters. Spearman rank correlation coefficients are described using the following scale: ±  very weak, ±  weak, ±  moderate, ±  strong, and ±  very strong. Cohort-wide descriptive statistics for hemodynamic stress parameters and geometric parameters are presented as mean ± standard deviation, unless otherwise stated. The coefficient of variation (CoV) was also used to describe the variation of a given parameter within the cohort. Two-sample One hundred and seventy nine subjects (358 carotid bifurcations) were segmented using the CNN and included in all analyses after 66 subjects were excluded based on a visual quality-assessment of the CE-MRA volume and the 4D Flow volumes, failed registrations or segmentations, or other acquisition errors. The majority of exclusions were a result of poor contrast-timing in the CE-MRA volume. Basic clinical characteristics, and geometric descriptions of the carotid bifurcation for this cohort are listed in Several hemodynamic parameters are shown in WSS and nwTKE at peak systole for example subjects from the lower, mid, and high tertiles are shown in The SA80 threshold values for TAWSS in the CCA, ECA, and ICA were 0.66, 0.72, and 0.68 [Pa], respectively. There was considerable spread in the amount of vessel surface area exposed to TAWSS below these thresholds, resulting in coefficients of variation within the cohort of 60, 60, and 53% for the CCA, ECA, and ICA, respectively. This indicates large interindividual variations in TAWSS patterns.The correlation coefficients for pairwise comparisons between the time-resolved parameters are listed in The correlation coefficients for pairwise comparisons between the non-temporally resolved parameters are listed in Correlation coefficients for pairwise comparisons between selected hemodynamic parameters  and geometric parameters  are listed in in vivo, and this study therefore offers a unique assessment of turbulent stresses on the vessel wall.This study characterized and compared several different expressions of hemodynamic shear stresses acting upon the vessel wall in a cohort of 179 subjects with asymptomatic carotid atherosclerosis. This study also explored the relationship between these shear stresses and the geometry of the carotid bifurcation. In addition, nwTKE was for the first time investigated Our results add evidence from a large cohort to the growing body of literature concerning MR-estimated WSS in the carotid bifurcation , 25–31 . While tWSS parameters displayed substantial variation within the cohort in our data. For example, with respect to TAWSS, the coefficients of variation were 32, 34, and 32% in the CCA, ECA, and ICA. However, this is in line with previously reported data and supports the conclusion that the carotid bifurcation has complex and variable flow patterns. For example, in branch-specific data from healthy volunteers, TAWSS coefficients of variation of 28, 32, and 35% in the CCA, ECA, and ICA were reported by Cibis et al. . In a suOur data yielded a similar correlation between OSI and TAWSS as seen in previous CFD-based work , though in vivo. Temporally resolved nwTKE and WSS were strongly correlated, ρ = 0.645, which can be expected as turbulence at the vessel wall to some degree develops near regions with high velocity and resulting flow detachment. The maximum nwTKE and maximum WSS values per branch were more strongly correlated (ρ = 0.642) than the minimum pairing (ρ = 0.460) at a given point in time. As visualized in In this study, nwTKE and WSS were examined together for the first time nwTKE provides information about the impact of disturbed flow on the vessel wall that is complementary to conventional assessment using OSI. OSI has been used as an indirect measurement for the effects of disturbed flow on the vessel wall as it measures the degree of flow reversal. While OSI represents laminar and periodic oscillations in WSS over the cardiac cycle, nwTKE represents rapid chaotic oscillations in WSS on the order of milliseconds or less. Indeed, we did not find strong correlations between OSI and TAnwTKE, in any branch, in our analyses. Similarly, TAnwTKE was not correlated to SA80, as expected given the strong relationship between SA80 and OSI (ρ = −0.690). This signals that nwTKE presents information that OSI does not, and therefore both should be examined to gain a more complete understanding of the stresses acting on the vessel wall.Vessel diameter and bifurcation angle displayed, at best, weak correlations to hemodynamic parameters in this study. Three whole-cycle parameters were examined: TAWSS, TAnwTKE, and OSI, as well as systolic WSS and systolic TKE. The strongest correlations were found between branch diameter and TAWSS, though these were still weak, and between OSI and the geometric parameters in the ECA. Our results therefore do not point to branch diameter or bifurcation angle alone as obvious or robust variables for predicting WSS or nwTKE, and add to previous research with similar findings . More coThis study has several limitations. It is well-known that MR-based WSS underestimates the true WSS due to poor spatial resolution. In addition, MRI-based measurements are subject to noise and segmentation errors that affect WSS estimates , 44, 47.in vivo investigations.In conclusion, this study estimated the hemodynamic wall stress parameters WSS, OSI, and nwTKE in a cohort of 358 carotid bifurcations and investigated the relationships between them as well as their relationships to basic geometric parameters. Correlations between WSS and nwTKE were moderate-to-strong for both their temporally resolved and cycle-averaged forms. In addition, nwTKE, reflecting the turbulent component of WSS, was not related to commonly used parameters for estimating the effects of disturbed flow such as the OSI and therefore appears to present new information. Hemodynamic parameters displayed weak correlations to vessel diameter and the bifurcation angle. WSS, OSI, and nwTKE exhibited substantial variation throughout the cohort, indicating that the degree and pattern of hemodynamic stresses acting on the vessel wall are not easily predicted and should be revealed using comprehensive The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This study was reviewed and approved by the Ethical Review Board at Umeå University . Participants provided their written informed consent to participate in this study.MZ, EdM, and PD conceived and designed the study. MZ, JA, EG, and PD analyzed the data. MZ, EG, JE, EdM, and PD interpreted the results. MZ and PD drafted the manuscript. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.