Met-enkephalin-induced release into the blood of a factor causing postural asymmetry

Met- and Leu-enkephalin applied subarachnoidally into the rostral portion of a transected spinal cord (at the T6-T7 level) induce postural asymmetry of the hind limbs in rats, Met-enkephalin being predominantly responsible for the flexion of the right, and Leu-enkephalin of the left, hind leg. The blood serum of rats injected with Met-enkephalin contains a factor which, when administered subarachnoidally into the caudal portion of the transected spinal cord, is capable of inducing the hind limb postural asymmetry--predominantly, with the right leg flexion. This factor is inactivated by papain and differs from Met- and Leu-enkephalin in chromatographic properties. Apparently, Met-enkephalin induces the release of a peptide factor into the blood, from the brain or organs innervated by the neurons lying above the cut. It is then carried with the blood to the hind limbs and effects the hind limb postural asymmetry.     

Opioids evoke postural asymmetry in rats: the side of the flexed paw depends on the type of opioid agonist

Opioid kappa-agonists bremazocine and dynorphin (1-13), sigma-agonist SKF 10.047 and delta-agonist D-Ala2, D-Leu5-enkephalin (DADL) induce postural asymmetry of rats hind limbs under subarachnoidal administration below the level of spinal cord section (T3-T4). The side of the flexed leg depends on the opioid agonist type: bremazocine and dynorphin (1-13) induce predominantly right flexion. SKF 10.047--the left flexion, but not in all doses, DADL--in small doses (1 and 100 pg per animal)--of the right one, in larger doses (up to 10 ng per animal)--of the left one. Saline and opiate mu-agonist morphine do not induce postural asymmetry. Opiate antagonist naloxone prevents asymmetry development when injected prior opioid agonists, and also decreases the number of asymmetries induced by these agonists. Naloxone alone does not influence the per cent of animals with pose asymmetry. The opioid receptors are involved in asymmetry development. The revealed ability of opioid kappa-, delta- and sigma-agonists may be based on lateralization of opioid receptors in the rat spinal cord.     

Mechanical hyperalgesia induced by endothelin-1 in rats is mediated via phospholipase C, protein kinase C, and MAP kinases

In addition to causing overt nociception, intraplantar (ipl) endothelin (ET)-1 injection into the rat hind paw induces hyperalgesia to mechanical stimuli, mediated via local ET(B) receptors coupled to protein kinase (PK) C, but not PKA. The present study further examines the intracellular signaling mechanisms underlying this effect of ET-1. ET-1 (30 pmol) or phospate-buffered saline (PBS) was injected ipl in rats and the threshold of responsiveness to mechanical stimulation was assessed repeatedly each hour up to 8 hrs and 24 hrs, using the dynamic plantar aesthesiometer test, which detects the minimal pressure required to evoke paw withdrawal. Different groups were treated, 15 mins before ET-1 administration, with ipsilateral injection of selective inhibitors of either phospholipase (PL) A2 (1 nmol PACOCF3), PLC (30 pmol U73122), PKC (1 nmol GF109203X), p38 mitogen-activated protein kinase (MAPK; 30 nmol SB203580), extracellular signal-regulated kinase (ERK1/2; 30 nmol PD98059), c-Jun N-terminal kinase (JNK; 30 nmol SP600125), or vehicle, to assess their influence on the hyperalgesic response. The mechanical hyperalgesia caused by ET-1 started 2 hrs after injection, peaked at 5 hrs (PBS, 29 +/- 0.5 g; ET-1, 17 +/- 1.3 g) and lasted up to 8 hrs. The inhibitors of PLC, PKC, p38 MAPK, ERK1/2, and JNK caused long-lasting reductions of the mechanical hyperalgesia (inhibitions at 4 hrs of 100%, 90%, 97%, 90%, and 100%, respectively), but the PLA2 inhibitor reduced hyperalgesia only at 4 hrs (by 58%). Thus, mechanical hyperalgesia triggered by ET-1 in the rat hind paw depends importantly on signaling pathways involving PLC, PKC, p38 MAPK, ERK1/2, and JNK, whereas the contribution of PLA2 is relatively minor.     

Chronopharmacological study of furosemide in rats: (II). Influence of beta-adrenoceptor blockade

We have previously demonstrated a time-dependent variability in the diuretic effect of furosemide in rats. The present study was undertaken to evaluate the influence of beta-adrenoceptor blockade on these time-dependent variations. Furosemide (5 mg/kg) was administered intra-arterially in Wistar rats at 1000 hrs (03HALO) or at 2200 hrs (15HALO) with pretreatment with either propranolol (10 mg/kg) or atenolol (10 mg/kg). Urine was collected for 60 min after furosemide administration and urinary excretion of sodium and furosemide were determined respectively. Propranolol pretreatment abolished the temporal variations observed in urine volume, urinary sodium and furosemide levels during the observation periods. With atenolol pretreatment, however, all these variables were significantly greater at 1000 hrs (03HALO) than at 2200 hrs (15HALO) as observed in the previous study. These results suggest that the beta-adrenoceptor-mediated stimuli, which is blocked by propranolol but not by atenolol, is responsible for the time-dependent changes in the diuretic effect of furosemide.     

Dynamics of the activity of the postural asymmetry factor after unilateral damage to the motor area of the cortex

The authors studied the time-course of functional rearrangements of the segmental apparatus after unilateral injury of the rat motor cortex. It was found that one day after injury the postural asymmetry of the hind limbs was fixed by the lumbal region of the spinal cord. This functional state of the segmental apparatus lasted 10 days after injury in the presence of the maximal activity of postural asymmetry factor (PAF) in the CSF and increasing activity of the factor in the brain tissue. Recovery of the segmental apparatus to symmetrical function by the end of the third week following injury was accompanied by PAF inactivation.     

Formation of postural asymmetry in rat hindlimbs during colchicine-induced unilateral blockade of axonal transport in cortico-lumbar projections

The possibility of the formation of spinal cord functional asymmetry by the blockade of axonal transport in corticolumbar projections with colchicine was investigated. To identify the blockade of axonal transport, the method of retrograde transport of horseradish peroxidase was used. The blockade of axonal transport led to the formation of the asymmetric functional status of the spinal cord, manifesting in postural asymmetry of the hind limbs and characteristic changes in the pattern of bioelectrical activity of the flexor muscles. An endogenous factor inducing postural asymmetry in intact recipients was detected in the cerebrospinal fluid of colchicine treated animals. Based on the experimental data the conclusion is drawn that interruption of normal axonal transport attests to the destruction of central neurons.     

Transplantation of Mesenchymal Cells Improves Peripheral Limb Ischemia in Diabetic Rats

Adipose tissue-derived mesenchymal stromal cells (ADSCs) are a prominent cellular source for regenerative medicine. We tested whether transplantation of ADSCs into the ischemic muscular tissue of diabetic animals would attenuate impaired cell metabolism and microcirculatory function. We induced unilateral hind limb ischemia in male streptozotocin-treated rats and nondiabetic controls. One day after femoral artery ligation, six rats per group were intramuscularly injected allogeneic ADSCs (10⁶–10⁷–10⁸ cells/mL); or conditioned media from ADSC cultures (CM); or saline; or allogeneic fibroblasts (10⁷ cells/mL); or nonconditioned medium. Rats underwent magnetic resonance angiography; short time inversion recovery (STIR) edema-weighed imaging; proton MR spectroscopy (¹H-MRS); immunoblotting and immunofluorescence on both hind limbs for 4 weeks. T₁-weighted and STIR images showed tissue swelling and signal hyperintensity, respectively, in the ischemic tissue. The mean total ratio of creatine/water for the occluded limbs was significantly lower than for the nonoccluded limbs in both nondiabetic and diabetic rats. ADSC and CM groups had greater recovery of tCr/water in ischemic limbs in both diabetic and nondiabetic rats, with increased expression of α-sarcomeric actinin, vascular endothelial growth factor and hepatocyte growth factor, as well as increased vessel density. ADSCs improve ischemic muscle metabolism and increase neovasculogenesis in diabetic rats.     

Chronopharmacological study of furosemide; (V). Influence of pretreatment with 6-hydroxydopamine

Our previous indirect evidences suggested that the adrenergic nervous system is involved in the mechanisms responsible for the time-dependent changes in the effects of furosemide in Wistar rats. In the present study, the role of this system was examined more directly by means of 6-hydroxydopamine-induced sympathectomy. Thirty mg/kg of 6-hydroxydopamine hydrobromide (6-OH-DA) (n = 9) or its vehicle alone (n = 9) was injected intra-arterially (i.a.) twice in Wistar rats. Furosemide (5 mg/kg) was administered i.a. at 1000 hrs (03HALO*) or at 2200 hrs (15HALO). Urine was collected for 60 min after the drug and urinary excretion of sodium and furosemide were determined respectively. Urine volume and urinary excretion of sodium and furosemide were significantly greater at 1000 hrs (03HALO) than at 2200 hrs (15HALO) in the vehicle-injected rats as observed in the previous study. However these administration-time-dependent changes in the effects of furosemide disappeared in the rats with 6-OH-DA. Thus, the present study provides more direct evidence and supports our original hypothesis concerning the mechanisms of this chronopharmacological phenomenon of the agent. Since 6-OH-DA does not penetrate the central nervous system from the blood stream, the present data also indicate that the peripheral adrenergic system is involved in this event.     

Heritabilities of Directional Asymmetry in the Fore- and Hindlimbs of Rabbit Fetuses

Directional asymmetry (DA), where at the population level symmetry differs from zero, has been reported in a wide range of traits and taxa, even for traits in which symmetry is expected to be the target of selection such as limbs or wings. In invertebrates, DA has been suggested to be non-adaptive. In vertebrates, there has been a wealth of research linking morphological asymmetry to behavioural lateralisation. On the other hand, the prenatal expression of DA and evidences for quantitative genetic variation for asymmetry may suggest it is not solely induced by differences in mechanic loading between sides. We estimate quantitative genetic variation of fetal limb asymmetry in a large dataset of rabbits. Our results showed a low but highly significant level of DA that is partially under genetic control for all traits, with forelimbs displaying higher levels of asymmetry. Genetic correlations were positive within limbs, but negative across bones of fore and hind limbs. Environmental correlations were positive for all, but smaller across fore and hind limbs. We discuss our results in light of the existence and maintenance of DA in locomotory traits.     

Effect of demedullation on freezing injury in hind limbs of rats

Freezing incidence and tissue loss on exposure of hind limbs of female Wistar rats to freezing mixture was reduced by demedullation 6 days prior to cold exposure (p<0.01 and p<0.001 respectively); demedullation 1 h after freezing injury had no effect on tissue loss. Noradrenaline (1 mg/kg i.p.) 5 min before exposure increased the freezing incidence in intact (p<0.05) as well as in demedullated rats (p<0.01), with no effect on tissue loss. Adrenaline (500 mg/kg i.p.) had no effect on either. A sustained fall in plasma adrenaline after demedullation leading to reduced reactivity of the blood vessels to some vasoactive agents is postulated.     

Chronic polyelectromyography in awake, unrestrained animals.

A procedure is described for making an implantable electrode array for recording EMG activity in muscles of awake, unrestrained animals (rats and cats) at rest, during rhythmic activity and in response to various reflexogenic stimuli. The electrode array consists of a percutaneous connector (covered with Dow-Corning Silastic Medical Adhesive), steel wire spiral leads contained in silicone tubing and silicone plate probes with platinum electrodes. These plate probes can be fixed either to the bone underneath the muscle, slipped under the fascia, or fixed between muscles. EMG records are presented of postural activity and ambulation in rats, mastication in cats and unilateral and bilateral spinal and supraspinal reflex responses in rat hind limb muscles up to 6 months after implantation. The advantages (and drawbacks) of this technique and its possible uses in neurophysiology are enumerated in the discussion.     

Effects of pargyline on hypothalamic biogenic amines and serum prolactin. LH and TSH in male rats.

The time course effects of pargyline on hypothalamic biogenic amines and serum prolactin (PRL), LH and TSH were studied in adult male rats. The rats were killed at intervals of 1–6 hrs after pargyline injection. Hypothalamic dopamine (DA) rose 79% by 1 hr and was 41% above “0” time by 6 hrs. Norepinephrine (NE) increased 31% by 1 hr and remained at about this level through 6 hrs, whereas serotonin (5HT) increased from 42% by 1 hr and to 95% by 6 hrs. Serum PRL LH and TSH fell significantly during the first 2 hrs, but all had returned to pretreatment values by 4 hrs. Serum PRL was about 4-fold above pretreatment values by 6 hrs, but LH and TSH remained at pretreatment levels. Stimulation by pargyline of PRL release was potentiated by Lilly compound 110140, a serotonin reuptake inhibitor, and blocked by parachlorophenylalanine, a serotonin synthesis inhibitor. These results suggest that the inhibitory effects of pargyline on PRL, LH, and TSH release during the first 2 hrs were associated mainly with a rapid increase in DA, and subsequent elevation of PRL release was related to the increase in 5HT. Return of serum LH and TSH to pretreatment levels at 4 and 6 hrs appeared to be associated mainly with the decrease in DA and perhaps to elevated NE levels. These results suggest that changes in relative concentrations of hypothalamic amines are related to differential release of PRL, LH and TSH.     

Effect of adrenalectomy and 5-hydroxytryptophan on phasic release of luteinizing hormone

The effect of 5-hydroxytryptophan (5-HTP) on serum progesterone and the possible role of adrenal progesterone in mediating stimulation by 5-HTP of phasic release of luteinizing. hormone (LH) were investigated in estradiol benzoate (EB)-treated ovariectomized rats. LH surges were induced in long-term (at least two weeks) ovariectomized rats by two injections of EB (20 micrograms/rat, s.c.) with an interval of 72 hrs. Administration of 5-HTP (50 mg/kg, i.p.) at 1000 hr in EB-treated ovariectomized rats resulted in a four-fold increase in serum progesterone within 30 mins, and significantly stimulated the LH surge at 1600 hr. This facilitative effect of 5-HTP on serum LH, but not progesterone, was further potentiated in rats pretreated with P-chlorophenylalanine (PCPA) 72 hrs earlier. Adrenalectomy shortly before 5-HTP administration attenuated the LH surge in saline treated controls, and completely blocked the facilitative effect of 5-HTP on the afternoon surge of LH in rats pretreated with PCPA 72 hrs earlier. On the other hand, chronic adrenalectomy (for 6 days) followed by hydrocortisone (0.2 mg/rat/day) replacement not only had no effect on the LH surge in saline treated controls, but also failed to prevent 5-HTP from facilitating the LH surge in PCPA pretreated rats. On the first day of bleeding, the basal LH value at 1000 hr in sham operated controls was significantly suppressed by PCPA pretreatment 48 hrs earlier. The second dose of 5-HTP administered on the next day failed to potentiate LH surges in either sham operated or adrenalectomized rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equine fetal kinetics: entry and retention of fetal hind limbs in a uterine horn

Transrectal ultrasonic examinations were made in 31 pregnant pony mares once a week during Months 6 to 11. Each uterine horn was divided into 3 approximately equal segments (caudal, middle, cranial). The percentage of examinations with cranial fetal presentation increased (P<0.05) progressively from 58% at Month 6 to 99% at Month 9 and was followed in all mares by entry of the fetal hind limbs into one uterine horn. The mean number of uterinehorn segments with limb parts increased (P<0.05) between each set of consecutive months from Month 6 to Month 10. Initially (Months 7 and 8), retraction of limbs after entry into the caudal and middle segments of the horn was common (31% incidence). The mean day of final entry of the limbs without detection of subsequent retraction was Day 230 +/- 2.2. Both uterine horns were closed during the examination preceding final entry of the hind limbs into one horn in 25 of 29 (86%) mares. The limbs reached the cranial segment in most examinations by Months 9 and 10 (73% and 98%). The cross-sectional height, as seen on the ultrasound screen, of both uterine horns increased (P<0.05) progressively during Months 7 to 10. Between Months 10 and 11, the height of the horn containing the limbs decreased (P<0.05); this result was attributable to a flattening of the horn and thinning (P<0.05) of the horn wall above a hoof and metatarsal bone. Apparent placental fluid (nonechogenic areas >5 mm in height) between the hind limbs and the cornual wall was detected in 0, 10, 25, and 63% (P<0.01) of examinations for Months 8 to 11, respectively. Results indicated that entry of the fetal hind limbs into a uterine horn was initially tentative and became final during a mean of Month 8. The apposition between the cornual wall and limbs was close during Months 7 to 11. By Month 11, the fetal-limb horn became flatter and thin-walled and usually contained placental fluid.     

Role of donor-specific regulatory T cells in long-term acceptance of rat hind limb allograft

Background

Vascularized bone marrow transplantation (VBMT) is widely accepted as an efficient means of establishing chimerism and inducing tolerance. However, the mechanism underlying is poorly understood. Recently, regulatory T cells (Tregs) have been shown to play an important role in regulating immune responses to allogeneic antigens. In this study, we explored the role of Tregs in the induction of tolerance in an allogeneic hind limb transplantation model.

Methodology/Principal Findings

Forty-eight Lewis rats were divided into 6 groups. They received isografts and allografts from Brown-Norway hind limbs. Recipients in groups 1 and 2 received isografts and those in the other groups received allografts. The bone components of donor limbs were kept intact in groups 1, 3, and 5 but removed before transplantation into groups 2, 4, and 6. Tapered cyclosporin A (CsA) was administered to recipients in groups 5 and 6 after transplantation. During the 100-day observation period, all isografts survived, but the allografts in groups 3 and 4 were rejected within 8 to 12 days. CsA-treated intact allografts survived rejection-free for more than 100 days, and CsA-treated allografts lacking bone elements were rejected within 2 months. Stable peripheral chimerism and myeloid chimerism were observed in group 5. Declining peripheral chimerism and a lack of myeloid chimerism were observed in group 6. Donor-specific Tregs were exclusively detected in both peripheral blood and in the spleens of long-term recipient rats in group 5, with an increased FoxP3 mRNA expression in the allografts. This was further demonstrated to be responsible for donor-specific hyporeactivity by in vitro one-way mixed lymphocyte reaction (MLR).

Conclusion/Significance

Bone components in the allogeneic hind limbs can induce myeloid chimerism and donor-specific Tregs may be essential to tolerance induction. The bone-removal hind limb model may be a suitable counterpart to the induction of tolerance in the study of limb transplantation.     

Challenging Postural Tasks Increase Asymmetry in Patients with Parkinson’s Disease

The unilateral predominance of Parkinson’s disease (PD) symptoms suggests that balance control could be asymmetrical during static tasks. Although studies have shown that balance control asymmetries exist in patients with PD, these analyses were performed using only simple bipedal standing tasks. Challenging postural tasks, such as unipedal or tandem standing, could exacerbate balance control asymmetries. To address this, we studied the impact of challenging standing tasks on postural control asymmetry in patients with PD. Twenty patients with PD and twenty neurologically healthy individuals (control group) participated in this study. Participants performed three 30s trials for each postural task: bipedal, tandem adapted and unipedal standing. The center of pressure parameter was calculated for both limbs in each of these conditions, and the asymmetry between limbs was assessed using the symmetric index. A significant effect of condition was observed, with unipedal standing and tandem standing showing greater asymmetry than bipedal standing for the mediolateral root mean square (RMS) and area of sway parameters, respectively. In addition, a group*condition interaction indicated that, only for patients with PD, the unipedal condition showed greater asymmetry in the mediolateral RMS and area of sway than the bipedal condition and the tandem condition showed greater asymmetry in the area of sway than the bipedal condition. Patients with PD exhibited greater asymmetry while performing tasks requiring postural control when compared to neurologically healthy individuals, especially for challenging tasks such as tandem and unipedal standing.     

Persistent pain model reveals sex difference in morphine potency

Central or systemic administration of agonists directed at the mu or delta opiate receptors generally produce a greater degree of analgesia in males than in females. To date, most studies examining sex-based differences in opioid analgesia have used acute noxious stimuli (i.e., tail-flick and hot plate test); thus the potential dimorphic response of centrally acting opiates in the alleviation of persistent inflammatory pain is not well established. In the present study, right hind paw withdrawal latency (PWL) to radiant thermal stimuli was measured in intact male and cycling female Sprague-Dawley rats before and after unilateral hind paw injection of the inflammatory agent complete Freund's adjuvant (CFA). Control animals received intraplantar injection of saline. Twenty four hours after CFA or saline injection, animals received either saline or morphine bisulfate (0.5-15 mg/kg sc). Separate groups of control or inflamed animals were tested on their responsiveness to morphine at 7, 14, and 21 days post-CFA or saline. No sex differences were noted for baseline PWLs, and females displayed slightly less thermal hyperalgesia at 24 h post-CFA. At all morphine doses administered, both the antihyperalgesic effects of morphine in the inflamed animals and the antinociceptive effects of morphine in control animals were significantly greater in males compared with females. Similarly, in males, the antihyperalgesic effects of morphine increased significantly at 7-21 days post-CFA; no significant shift in morphine potency was noted for females. These studies demonstrate sex-based differences in the effects of morphine on thermal hyperalgesia in a model of persistent inflammatory pain.     

Behavioral correlates of serotonin depletion.

Depletion of telencephalic serotonin (5-HT) content by medical forebrain bundle lesions, which interrupt the ascending serotonergic pathways or by DL-p-chlorophenylalanine produces an increased sensitivity to pain as measured by the flinch-jump, stabilimetric, or hot-plate methods. Examination of the effects of a number of other lesions and drugs indicated that dopamine, norepinephrine and acetylcholine are not involved in pain sensitivity. Dosages of 75 mg/kg DL-5-hydroxytryptophan(5-HTP), 37.5 mg/kg L-5-HTP or 50 mg/kg Ro 4-4602 (NI-(DL-seryl)-N2-(2,3,4-trihydroxybenzyl)hydrazine) plus 37.5 mg/kg L-5-HTP administered to medical forebrain bundle lesioned rats returned both the telencephalic content of 5-HT and the pain threshold to normal values. Injection of 37.5 mg/kg of D-5-HTP or an equimolar dose of L-dopa had no effect on pain threshold. Normal animals display increased sensitivity to pain and decreased 5-HT contents in frontal pole, hippocampus, and amygdala during dark as compared to light hours. All three of these telencephalic areas are innervated by the ascending serotonergic pathways, and cells in these areas show inhibition of firing following the iontophoretic application of 5-HT. Taken together these data suggest that the serotonergic system normally acts to inhibit the effects of painful stimuli. A review of a variety of behavioral effects of 5-HT depletion including an enhanced response to lysergic acid diethylamide and amphetamine suggests that the ascending serotonergic system may have a general role in the inhibition of arousal, rather than a specific role with respect to various categories of behavior.     

The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia

After a dose of 10 micrograms of 6-aminonicotinamide (6-AN) was administered to day-4- chick embryo in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis. The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [35S]sulfate and [3H]glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the 35S/3H ratio, the proportion of unsulfated disaccharide (delta Di-0S), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of 35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilage-specific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation. We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder, i.e., micromelia.     

Analgesic,anti-inflammatory and anti-ulcer properties of Thai Perilla frutescence fruit oil in animals

Perilla frutescens fruit oil (PFO) is rich in α-linolenic acid (ALA) and exhibits biological activities. We aimed to investigate analgesic, anti-inflammatory and anti-ulcer activities of PFO and PFO-supplemented soybean milk (PFO-SM) in animal models. Analgesic activity was assessed in acetic acid-induced writhing in mice, while anti-inflammatory activity was performed in ethyl phenylpropiolate (EPP)-induced ear edema and carrageenan-induced hind paw edema in rats. Anti-ulcer effects were conducted in water immersion stress, HCl/ethanol and indomethacin-induced gastric ulcer in rats. Distinctly, PFO, containing 6.96 mg ALA and 2.61 mg LA equivalence/g, did not induce acute toxicity (LD₅₀ > 10 mL/kg) in mice. PFO (2.5 and 5 mL/kg) and PFO-SM (0.05 mL PFO equivalence/kg) inhibited incidences of writhing (16.8, 18.0 and 32.3%, respectively) in acetic acid-induced mice. In addition, topical applications of PFO (0.1 and 1 mL/ear) significantly inhibited EPP-induced ear edema (59.3 and 65.7%, respectively) in rats, while PFO-SM slightly inhibited ear edema (25.9%). However, PFO and PFO-SM did not inhibit carrageenan-induced hind paw edema in rats. Indeed, PFO (2.5 and 5 mL/kg) significantly inhibited gastric ulcers in rats that induced by water immersion stress (92.4 and 96.6%, respectively), HCl/ethanol (74.8 and 73.3%, respectively) and indomethacin (68.8 and 88.9%, respectively), while PFO-SM did not. PFO displayed potent analgesic, anti-inflammatory and anti-ulcer properties, while PFO-SM exerted only analgesic properties. Thus, Thai PFO and its functional drink offer potential benefits in treatment of analgesic, inflammatory diseases and gastric ulcer.