Subcutaneous lysophosphatidylcholine administration promotes a febrile and immune response in Holstein heifer calves.

Lysophosphatidylcholine (LPC) is immunomodulatory in nonruminants; however, the actions of LPC on immunity in cattle are undefined. Our objective was to study the effects of LPC administration on measures of immunity, liver health, and growth in calves. Healthy Holstein heifer calves (n = 46; age 7 ± 3 d) were randomly assigned to 1 of 4 treatments (n = 10 to 11 calves/treatment): a milk replacer diet unsupplemented with lecithin in the absence (CON) or presence of subcutaneously (s.c.) administered mixed (mLPC; 69% LPC-16:0, 25% LPC-18:0, 6% other) or pure LPC (pLPC; 99% LPC-18:0), or a milk replacer diet supplemented with 3% lecithin enriched in lysophospholipids containing LPC in the absence of s.c.-administered LPC (LYSO) for 5 wk. Calves received 5 s.c. injections of vehicle (10 mL of phosphate-buffered saline containing 20 mg of bovine serum albumin/mL; CON and LYSO) or vehicle containing mLPC or pLPC to provide 10 mg of total LPC per kilogram of BW per injection every 12 h during wk 2 of life. Calves were fed a milk replacer containing 27% crude protein and 24% fat at 1.75% of BW per day (dry matter basis) until wk 6 of life (start of weaning). Starter grain and water were provided ad libitum. Body measurements were recorded weekly, and clinical observations were recorded daily. Blood samples were collected weekly before morning feeding and at 0, 5, and 10 h, relative to the final s.c. injection of vehicle or LPC. Data were analyzed using a mixed model, with repeated measures including fixed effects of treatment, time, and their interaction. Dunnett's test was used to compare treatments to CON. Peak rectal temperatures were higher in mLPC or pLPC, relative to CON. Plasma LPC concentrations were greater in mLPC and LYSO calves 5 h and 10 h after the final injection, relative to CON. Calves receiving mLPC and pLPC also had higher circulating serum amyloid A concentrations, relative to CON. Calves receiving mLPC had greater serum aspartate aminotransferase, γ-glutamyltransferase, and glutamate dehydrogenase concentrations, relative to CON. Calves provided mLPC experienced lower average daily gain (ADG) after weaning, relative to CON. The LYSO treatment did not modify rectal temperatures, ADG, or measures of liver health, relative to CON. We conclude that LPC administered as s.c. injections induced an acute febrile response, modified measures of liver and immune function, and impaired growth in calves.

Effects of dietary organic acid and pure botanical supplementation on growth performance and circulating measures of metabolic health in Holstein calves challenged by heat stress.

To evaluate the effects of heat stress environmental conditioning and dietary supplementation with organic acid and pure botanicals (OA/PB) on growth in dairy calves, we enrolled 62 bull (noncastrated) and heifer calves in a study with a completely randomized design. Calves were assigned to 1 of 5 groups (n = 11 to 14/group): (1) thermoneutral conditions (TN-Con), (2) HS conditions (HS-Con), (3) thermoneutral conditions and pair-fed to match nutrient intake with HS-Con (TN-PF), (4) HS with low-dose OA/PB [75 mg/kg of body weight (BW); 25% citric acid, 16.7% sorbic acid, 1.7% thymol, 1.0% vanillin, and 55.6% triglyceride; HS-Low], or (5) HS with high-dose OA/PB (150 mg/kg of BW; HS-High). Supplements were delivered as a twice-daily bolus via the esophagus from wk 1 through 13 of life; all calves, including those on the control treatments, received an equivalent amount of triglyceride used for microencapsulation. Calves were raised in TN conditions from birth until weaning. After weaning, calves (62 ± 2 d; 91 ± 10.9 kg of BW) were transported to a new facility and remained in TN conditions [temperature-humidity index (THI): 60 to 69] for a 7-d covariate period. Thereafter, calves remained in TN or were moved to HS conditions (THI: diurnal change 75 to 83 during night and day, respectively) for 19 d. Clinical assessments were performed thrice daily, BW was recorded weekly, and blood was sampled on d 1, 2, 3, 8, 15, and 19. Upon experiment completion, calves from HS-Con and TN-Con were euthanized, and hot carcass and visceral organ weights were recorded. The mixed model included calf as a random effect; treatment, day, hour (when appropriate) as fixed effects, and the interactions of treatment × day and treatment × hour (when appropriate). Rectal and skin temperatures and respiration rates were greater in HS-Con than in TN-Con. During heat stress exposure, dry matter intake (DMI), average daily gain (ADG), and gain to feed (G:F) were lower in HS-Con relative to TN-Con. Comparing HS-Con and TN-PF, ADG and G:F were similar. Plasma fatty acid concentrations were elevated in TN-PF compared with HS-Con and TN-Con. Despite tendencies for increased aspartate aminotransferase, HS conditions did not overtly influence liver and inflammation markers. Liver weights were lower in HS-Con relative to TN-Con. During the first week of heat exposure, DMI was greater for HS-Low relative to HS-Con. Supplementation of OA/PB at low and high levels had a similar G:F to HS-Con. We conclude that reductions in DMI accounted for production losses during HS conditioning and that dietary OA/PB supplementation was not able to improve growth performance in heat-stressed calves.

Effects of dietary deoiled soy lecithin supplementation on milk production and fatty acid digestibility in Holstein dairy cows.

Deoiled soy lecithin is a feed additive enriched in phospholipids. Our study evaluated the effects of dietary deoiled soy lecithin supplementation on (1) milk production and composition, (2) plasma and milk fatty acid (FA) content and yield, and (3) apparent FA digestibility and absorption in lactating dairy cows fed fractionated palm fat. In a split-plot Latin square design, 16 Holstein cows (160 ± 7 days in milk; 3.6 ± 1.2 parity) were randomly allocated to a main plot receiving a corn silage and alfalfa haylage-based diet with palm fat containing either moderate (MPA) or high palmitic acid (HPA) content at 1.75% of ration dry matter (72 or 99% palmitic acid, respectively; n = 8/palm fat diet). On each palm fat diet, deoiled soy lecithin was top-dressed at 0, 0.12, 0.24, or 0.36% of ration dry matter in a replicated 4 × 4 Latin square design. Following a 14-d covariate period, lecithin supplementation spanned 14 d, with milk and blood collected during the final 3 d. Milk composition and pooled plasma markers were measured. The statistical model included the fixed effects of palm fat type, lecithin dose, period, and the interaction between palm fat type and lecithin dose. The random effect of cow nested within palm fat group was also included. Lecithin linearly decreased dry matter intake. In cows fed HPA, lecithin feeding reduced milk fat content and tended to decrease milk fat yield. Although no changes in milk yield were observed, a quadratic reduction in 3.5% fat-corrected milk was observed with increasing lecithin dose. Lecithin linearly increased energy-corrected milk efficiency in cows fed MPA. Lecithin supplementation also decreased milk urea nitrogen, relative to unsupplemented cows. The proportion of 16-carbon FA in milk fat decreased linearly with lecithin dose, whereas 18-carbon FA increased linearly. Lecithin reduced de novo FA (<16-carbon) content and tended to increase preformed FA (>16-carbon) content in a linear manner. Compared with MPA, HPA diets reduced apparent total and 16-carbon FA digestibility and absorption. Deoiled soy lecithin feeding did not modify FA digestibility or absorption. Our observations suggest that soy lecithin feeding modifies rumen digestion to reduce dry matter intake and change milk composition.

Effects of abomasal infusions of fatty acids and one-carbon donors on hepatic ceramide and phosphatidylcholine in lactating Holstein dairy cows.

Our objectives were to (1) determine whether the abomasal infusion of behenic acid (C22:0) elevated hepatic ceramide relative to palmitic acid (C16:0) or docosahexaenoic acid (C22:6n-3) infusion; (2) assess whether the abomasal infusion of choline chloride or l-serine elevated hepatic phosphatidylcholine (PC) in cows abomasally infused with C16:0; and (3) characterize the PC lipidome in cows abomasally infused with C22:6n-3, relative to C16:0 or C22:0 infusion. In a 5 × 5 Latin square design, 5 rumen-cannulated Holstein cows (214 ± 4.9 DIM; 3.2 ± 1.1 parity) were enrolled in a study with 6-d periods. Abomasal infusates consisted of (1) palmitic acid (PA; 98% C16:0); (2) PA + choline chloride (PA+C; 50 g/d choline chloride); (3) PA + l-serine (PA+S; 170 g/d l-serine); (4) behenic acid (BA; 92% C22:0); and (5) an algal oil rich in docosahexaenoic acid (DHA; 44% C22:6n-3). Emulsion infusates provided 301 g/d of total fatty acids containing a minimum of 40 g/d of C16:0. Cows were fed a corn silage-based diet. Milk was collected on d -2, -1, 5, and 6. Blood was collected and liver biopsied on d 6 of each period. Although we did not detect differences in milk yield, milk fat yield and content were lower in cows infused with DHA relative to PA. Plasma triacylglycerol concentrations were lower with DHA treatment relative to PA or BA. Cows infused with DHA had lower plasma insulin concentrations relative to cows infused with PA only. For objective 1, hepatic ceramide-d18:2/22:0 was highest in cows infused with BA relative to other treatments. For objective 2, plasma free choline concentrations were greater in PA+C cows relative to PA; however, we did not observe this effect with PA+S. Plasma total PC concentrations were similar for all treatments. Regarding the hepatic lipidome, a total of 18 hepatic PC were higher (e.g., PC-16:1/18:2) and 25 PC were lower (e.g., PC-16:0/22:6) with PA+C infusion relative to PA. In addition, 17 PC were higher (e.g., PC-20:3/22:5) and 21 PC were lower (e.g., PC-18:0/22:6) with PA+S infusion relative to PA. For objective 3, hepatic concentrations of many individual saturated PC (e.g., PC-18:0/15:0) were lower with DHA relative to other treatments. Hepatic concentrations of highly unsaturated PC with very-long-chain fatty acids (e.g., PC-14:0/22:6) were higher in DHA-infused cows relative to PA, PA+C, PA+S, or BA. The abomasal infusion of emulsions containing palmitic acid, palmitic acid with choline chloride or serine, behenic acid, or docosahexaenoic acid influence the hepatic ceramide and PC profiles of lactating cows.

Use of multiple admission variables better predicts intestinal strangulation in horses with colic than peritoneal or the ratio of peritoneal:blood l-lactate concentration.

BACKGROUND: Early identification of strangulating obstruction (SO) in horses with colic improves outcomes, yet early diagnosis of horses requiring surgery for SO often remains challenging. OBJECTIVES: To compare blood and peritoneal fluid l-lactate concentrations, peritoneal:blood l-lactate ratio, peritoneal minus blood (peritoneal-blood) l-lactate concentration and other clinical variables for predicting SO and SO in horses with small intestinal lesions (SO-SI) and then to develop a multivariable model to predict SO and SO-SI. STUDY DESIGN: Retrospective cohort. METHODS: A total of 197 equids admitted to a referral institution for colic between 2016 and 2019 that had peritoneal fluid analysis performed at admission were included. Twenty-three admission variables were evaluated individually for the prediction of a SO or SO-SI and then using multivariable logistic regression. Odds ratios (ORs) with 95% confidence intervals (CI) and area under the curve of the receiver operator characteristic (AUC ROC) were calculated. RESULTS: All variables performed better in the model than individually. The final multivariable model for predicting SO included marked abdominal pain (OR 5.31, CI 1.40-20.18), rectal temperature (OR 0.30, CI 0.14-0.64), serosanguineous peritoneal fluid (OR 35.34, CI 10.10-122.94), peritoneal-blood l-lactate (OR 1.77, CI 1.25-2.51), and peritoneal:blood l-lactate ratio (OR 0.36, CI 0.18-0.72). The AUC ROC was 0.91. The final multivariable model for predicting SO-SI included reflux volume (OR 0.69, CI 0.56-0.86), blood l-lactate concentration (OR 0.43, CI 0.22-0.87), serosanguineous peritoneal fluid (OR 4.99, CI 1.26-19.74), and peritoneal l-lactate concentration (OR 3.77, CI 1.82-7.81). MAIN LIMITATIONS: Retrospective, single-hospital study design. CONCLUSIONS: Blood and peritoneal fluid l-lactate concentrations should be interpreted in conjunction with other clinical variables. The relationship between peritoneal and blood l-lactate concentration for predicting SO or SO-SI was complex when included in a multivariable model. Models to predict SO probably vary based on lesion location.

Perioperative analgesic effects of an ultrasound-guided transversus abdominis plane block using bupivacaine in goats undergoing celiotomy.

Introduction: Never has the anatomy, the procedure of the transversus abdominis plane (TAP) block, or the perioperative analgesic effects of a bupivacaine TAP block been described in goats. Methods: This report details the relevant anatomy in a cadaveric study combined with the description/use of a TAP block in a controlled, randomized, prospective, blinded clinical study in which 20 goats with urolithiasis presenting for either ventral midline or paramedian celiotomy were enrolled. Anesthesia was induced with ketamine and midazolam and maintained with desflurane in oxygen. An ultrasound-guided TAP block was performed using 0.25% bupivacaine (4 sites, 0.4 mL/kg each site) (bupivacaine-TAP, n = 10) or equal volume of saline (control-TAP, n = 10). When indicated, urethral amputation was performed followed by celiotomy with cystotomy or tube cystostomy. Urethrotomy was performed if warranted. Intraoperatively, a 20% increase in mean arterial pressure (MAP), heart rate (HR) and/or respiratory frequency was treated with an increase in desflurane concentration of 0.5 Vol.%. Goats received ketamine boluses (0.2 mg/kg IV) when moving spontaneously. At 2, 12, and 24 h post-extubation, pain was scored with a descriptive scale. Data were analyzed with an analysis of variance (ANOVA) or the Wilcoxon signed-rank test, and P < 0.05 was considered statistically significant. Results: Bupivacaine-TAP goats exhibited lower end-tidal desflurane concentration requirements (P = 0.03), lower pain scores at 2-h post-extubation (P = 0.02), shorter anesthetic recovery times (P = 0.03) and decreased HR and MAP during surgical stimulation. Goats receiving a bupivacaine TAP block experienced less intraoperative nociceptive input requiring less inhalant anesthetic leading to faster anesthetic recoveries and decreased postoperative pain. Discussion: Ultrasound-guided TAP block is a simple technique to decrease anesthetic requirement while providing additional postoperative comfort in goats undergoing celiotomy.

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training.

Three dimensional models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Somatotropin increases plasma ceramide in relation to enhanced milk yield in cows.

Recombinant bovine somatotropin (rBST) changes metabolism to spare glucose for milk synthesis in cows. Ceramides inhibit insulin responsiveness in bovine adipocytes and are associated with insulin resistance and milk production in cows. The mechanisms by which rBST supports lactation may involve ceramide. Eight multiparous lactating Holstein cows were enrolled in a 2 × 2 replicated Latin square design with 14-d periods. Cows received a single rBST injection (Posilac; Elanco Animal Health, Indianapolis, IN; 0.062 mg/kg BW) or no injection (CON). An epinephrine challenge, insulin tolerance test, and liver biopsy were performed. Somatotropin enhanced the conversion of feed nutrients into milk components and increased plasma free fatty acid (FFA) concentrations (P < 0.01). Area-under-the-curves for FFA in response to epinephrine and insulin were greater in rBST-treated cows. In response to insulin, glucose concentrations (20- and 30-min post-challenge) and insulin area-under-the-curve were higher with rBST treatment (P < 0.05, <0.10, and <0.01), suggesting insulin resistance. Somatotropin modified the plasma lipidome. For example, rBST decreased plasma di- and triacylglycerol levels (eg, DG-50:1 and TG-18:0/16:0/16:1), phosphatidylcholines and sphingomyelins (P < 0.05). Somatotropin increased plasma total and very-long-chain (C22:0-, C24:0-, C26:0-) ceramide concentrations (P < 0.01). Liver ceramide concentrations were not modified. Plasma ceramides were positively correlated with circulating FFA (r ~ 0.57; P < 0.05) and milk yield (r ~ 0.63; P < 0.05). We conclude that rBST administration modifies the bovine lipidome and increases plasma ceramide concentrations in association with increased milk production in cows.

Clinical Assessment of an Ipsilateral Cervical Spinal Nerve Block for Prosthetic Laryngoplasty in Anesthetized Horses.

The nociceptive blockade of locoregional anesthesia prior to surgical stimulation can decrease anesthetic agent requirement and thereby potential dose-dependent side effects. The use of an ipsilateral second and third cervical spinal nerve locoregional anesthetic block for prosthetic laryngoplasty in the anesthetized horses has yet to be described. Anesthetic records of 20 horses receiving locoregional anesthesia prior to laryngoplasty were reviewed and compared to 20 horses of a similar patient cohort not receiving locoregional anesthesia. Non-blocked horses were 11 times more likely to require adjunct anesthetic treatment during surgical stimulation (P = 0.03) and were 7.4 times more likely to receive partial intravenous anesthesia in addition to inhalant anesthesia (P = 0.01). No horse in the blocked group received additional sedation/analgesia compared to the majority of non-blocked horses (75%) based on the anesthetist's perception of anesthetic quality and early recovery movement. No difference in recovery quality was observed between groups (P > 0.99). Cervical spinal nerve locoregional anesthesia appears well-tolerated and useful in reducing cumulative anesthetic agent requirement and may decrease the need for additional sedation/analgesia in horses undergoing anesthetized prosthetic laryngoplasty.

Cervical Epidural and Subarachnoid Catheter Placement in Standing Adult Horses.

Horses underwent either cervical epidural space (CES) catheterization or subarachnoid space (SAS) catheterization while restrained in stocks, under deep sedation (detomidine and morphine) and local anesthesia (mepivacaine 2%) block. Catheters were placed under ultrasound guidance with visualization of the dura, SAS, and spinal cord between the first (C1) and second (C2) cervical vertebrae. Following sedation and sterile skin preparation, operator 1 placed under ultrasound guidance, a 6- or 8-inch Tuohy needle with the bevel oriented caudally. For CES, a 6-inch Touhy needle was used with the hanging drop technique to detect negative pressure, and operator 2 then passed the epidural catheter into the CES. For SAS, following puncture of the dura, cerebrospinal fluid (CSF) was aspirated prior to placement of the epidural catheter. Placement into either CES or SAS was confirmed with plain and contrast radiography. Catheters were wrapped for the duration of the study. CSF cytology was assessed up to every 24 h for the study period. Horses were assessed daily for signs of discomfort, neck pain, catheter insertion site swelling, or changes in behavior. A complete postmortem assessment of the spinal tissues was performed at the end of the study period (72 h). Two horses had CES catheters and five horses had SAS catheters placed successfully. All horses tolerated the catheter well for the duration of the study with no signs of discomfort. Ultrasound was essential to assist placement, and radiography confirmed the anatomical location of the catheters. CSF parameters did not change over the study period (P > 0.9). There was evidence of mild meningeal acute inflammation in one horse and hemorrhage in another consistent with mechanical trauma. Placement of an indwelling CES or SAS catheter appears to be safe, technically simple, and well tolerated in standing sedated normal horses.

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

Rollers Versus Trainers: 10-Km Time Trial.

The primary aim of this investigation was to determine which cycling training device, Rollers or Trainers, was most effective in improving 10-km time trial. Eight male and 6 female volunteers (N = 14; age = 23.6 ± 4.6 yrs; height = 172.7 ± 9.9 cm; body mass = 68.4 ± 10.4 kg; % body fat = 16.9 ± 7.7; VO2max = 61.0 ± 9.4 ml·kg-1·min-1) provided informed consent prior to participation. Participants performed a10-km time trial at baseline and were then randomly assigned into one of three groups: Rollers (R), Trainers (T), or Control (C). Participants assigned to the R or T groups attended 24 supervised workout sessions throughout an 8-wk period (F: 3 days/week; I: 65-80% HRmax; D: 40 min; M: R or T). There were no significant differences in baseline 10-km time trial between R, T, and C groups [F(2,12) = 0.34, p = .72]. There was a significant difference in 10-km time trial improvement between groups post-assessment when controlling for baseline values (F = 17.04, p <.001). R participants improved by 20.4s [t(4) = 4.86, p = .008] and T participants improved by 12.8s [t(4) = 4.57, p = .01], while there was no significant improvement for subjects in C. Participants using R and T displayed significant decrements in time with respect to the 10-km time trial. However, R had a greater improvement in 10-km time trial when compared to T.

Thrombin activation of human platelets causes tyrosine phosphorylation of PLC-gamma 2.

Human platelets contain phospholipase C (PLC)-gamma 2, a distinct isoform closely related to PLC-gamma 1. Both inositol phospholipid-specific phospholipases C contain the src-related SH2 regions. Stimulation of platelets with the potent agonist, thrombin, led to a rapid and transient phosphorylation of PLC-gamma 2 on tyrosine residues. Activated platelets lysed in the absence of sodium orthovanadate had levels of tyrosine-phosphorylated PLC-gamma 2 paralleling those seen in unstimulated platelets. Previously, it had been shown that PLC-gamma 1 was phosphorylated on tyrosine residues by the agonist-occupied platelet-derived growth factor (PDGF) receptor and epidermal growth factor (EGF) receptor in cells other than platelets. In addition, more recent data have indicated that PLC-gamma 2 is also capable of being tyrosine-phosphorylated in cells of hematopoietic origin, such as B cells and natural killer (NK) cells. Here we report that PLC-gamma 2 expressed in a terminally-differentiated hematopoietic cell is also tyrosine-phosphorylated in response to an agonist.

The discovery of 9-cis retinoic acid: a hormone that binds the retinoid-X receptor.

Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid-X receptors (RXRs), mediate the physiologic activity of retinoids. The RXRs can form biologically active heterodimers with the RARs and with other nuclear receptors, including the vitamin-D, thyroid hormone, and peroxisome proliferator-activated receptors. Thus, the RXRs may play a pivotal role in modulating the action of other hormones or ligands. The RXRs were originally classified as orphan receptors whose cognate ligand was unknown until recently, when 9-cis retinoic acid (9-cis RA) was discovered to bind directly and activate this family of receptors. Since 9-cis RA also binds and activates the RARs, it is interesting to speculate that this natural ligand may regulate a broad range of physiologic processes by mediating transcriptional activity through both RAR- and RXR-linked pathways.

An approach to studying circadian rhythms of adolescent humans.

The "long nights" protocol was designed to evaluate sleep processes and circadian rhythm parameters in young humans. A total of 19 children (10 boys, ages 11.2 to 14.1 years [mean = 12.7 +/- 1.0], and 9 girls, ages 12.2 to 14.4 years [mean = 13.1 +/- 0.7]) took part in the study. Sleep/wake initially was assessed at home using actigraphy and diary for 1 week on each child's self-selected schedule followed by an 8-night fixed light-dark (LD) condition, while sleeping from 22:00 to 08:00 h and wearing an eye mask to exclude as much light as possible. Phase measurements included 4-night mean actigraphically estimated sleep onset and offset as well as 1-night dim light salivary melatonin onset (DLSMO) phase at the end of each condition. Subjects then lived in the laboratory for 6 consecutive cycles: Day 1 LD = 14:10 h, lights out 22:00 to 08:00 h; Days 2-4 LD = 6:18 h, lights out 18:00 to 12:00 h; Days 5-6 = constant routine in continuous dim light (about 20 lux); Night 6 = 14 h recovery sleep. Phase markers (sleep onset, sleep offset, DLSMO) were significantly less dispersed after the fixed LD as compared to the self-selected condition, indicating efficacy of the LD protocol. Phase markers were correlated at the self-selected assessment (sleep onset vs. sleep offset r = .72; DLSMO vs. sleep onset r = .82; DLSMO vs. sleep offset r = .76) but not on the fixed schedule, probably due to restricted range. The constant routine provided additional phase markers, melatonin offset and midphase. Offset phase of melatonin secretion was significantly correlated with age (r = .62) and Tanner stage (r = .62). In conclusion, these preliminary data indicate a relationship between adolescent development and circadian phase. Thus, the long nights protocol is a feasible way in which to assess circadian parameters in young humans as well as to examine intrinsic sleep processes.

Pathologic evaluation of the human suprachiasmatic nucleus in severe dementia.

Sleep disruption and other circadian rhythm disturbances are frequently seen in dementia patients. In this study, we examined the suprachiasmatic nucleus (SCN), the putative site of the hypothalamic circadian pacemaker, to determine the nature and degree of pathologic changes caused by severe dementia. Neuropathologic examination indicated that among 30 patients with a clinical history of severe dementia, 22 had Braak and Braak stage V-VI Alzheimer disease, 3 had combined Alzheimer and Parkinson disease, 3 had Pick disease and 2 had severe hippocampal sclerosis. Comparisons were made with a control group composed of 13 age-matched patients with no clinical or pathological evidence of dementia or other CNS disorders. To determine the pathologic involvement within the SCN, human hypothalami were stained with: Nissl, Bielchowsky silver, thioflavin S and specific antibodies directed against vasopressin (VP), neurotensin (NT), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), beta-amyloid (B/A4) and glial fibrillary acidic protein (GFAP). Pathologic damage was primarily limited to neuronal loss and neurofibrillary tangle formation. Only rare diffuse plaques were noted. The pathologic changes within the SCN were less severe than in the other brain regions. Morphometric analysis was accomplished using a stereological approach to sample the average total number of positively stained neurons and astrocytes in 10 different 0.1mm2 microscopic fields in the dorsal subdivision of the SCN. Patients with Alzheimer disease exhibited a significant decrease in vasopressin (9.75 vs 16.7, p < 0.001) and neurotensin (6.82 vs 9.63, p < 0.002) neurons, as well as a corresponding increase in the GFAP-stained astrocyte/Nissl-stained neuron ratio (0.54 vs 0.10, p < 0.009). These studies provide evidence that both vasopressin and neurotensin neurons are lost in Alzheimer disease, and that the astrocyte/neuron ratio is a reliable indicator of disease-related pathology within the SCN. Taken collectively, our data support the hypothesis that damage to the SCN may be an underlying anatomical substrate for the clinically observed changes in circadian rhythmicity that have been observed in Alzheimer patients.

Indomethacin reverses the microglial response to amyloid beta-protein.

Alzheimer's disease (AD) brains display intense microglial immunoreactivity in the area of senile plaques, suggesting that amyloid beta-protein may stimulate microglial infiltration. The activated microglia may modulate an immune response in the brain. Non-steroidal anti-inflammatory drugs (NSAIDs) are candidate therapeutics for AD because their effects on immune system components may influence the course of the disease. The present study examined the effects of an NSAID (indomethacin) on amyloid beta-protein-induced microglial infiltration. Amyloid beta-protein was chronically infused into rat lateral ventricles for 2 weeks. Extracellular amyloid beta-protein deposited along the lining and diffused into the tissue surrounding the lateral ventricle. Immunocytochemical staining showed that animals receiving amyloid beta-protein exhibited dramatic microglial response when compared to vehicle-infused rats. Activated microglia surrounded immunopositive amyloid beta-protein deposits, but this response was significantly attenuated in animals receiving either concurrent i.c.v. or subcutaneous (s.c.) treatment with indomethacin. These results suggest that chronic amyloid beta-protein infusion induces the proliferation of activated microglia and that indomethacin may be an effective treatment for inhibiting microglial proliferation.

Ventrolateral preoptic nucleus contains sleep-active, galaninergic neurons in multiple mammalian species.

The ventrolateral preoptic nucleus (VLPO) is a group of sleep-active neurons that has been identified in the hypothalamus of rats and is thought to inhibit the major ascending monoaminergic arousal systems during sleep; lesions of the VLPO cause insomnia. Identification of the VLPO in other species has been complicated by the lack of a marker for this cell population, other than the expression of Fos during sleep. We now report that a high percentage of the sleep-active (Fos-expressing) VLPO neurons express mRNA for the inhibitory neuropeptide, galanin, in nocturnal rodents (mice and rats), diurnal rodents (degus), and cats. A homologous (i.e. galanin mRNA-containing cell group) is clearly distinguishable in the ventrolateral region of the preoptic area in diurnal and nocturnal monkeys, as well as in humans. Galanin expression may serve to identify sleep-active neurons in the ventrolateral preoptic area of the mammalian brain. The VLPO appears to be a critical component of sleep circuitry across multiple species, and we hypothesize that shrinkage of the VLPO with advancing age may explain sleep deficits in elderly humans.

Hormonal and pharmacological manipulation of the circadian clock: recent developments and future strategies.

The mammalian circadian oscillator, located in the suprachiasmatic nuclei of the anterior hypothalamus, serves as the principal source of rhythmic temporal information for virtually all physiologic processes in the organism, including the alternating expression of sleep and wakefulness. Recent studies, in both animal models and human subjects, have demonstrated the important modulation of sleep and wakefulness mediated by the circadian clock. Independent of other factors, notably prior sleep-wake history, the circadian clock potentiates wakefulness (and alertness) at one phase of the diurnal cycle, while facilitating sleep and its attendant processes at the opposite phase. The adaptive advantage of synchronizing sleep-wake behaviors with the daily changes in the external environment is clear. But in a modern world where the constraints of environmental time are less and less important, the circadian clock still imposes rigid boundaries on the timing of sleep and alert wakefulness that are increasingly perceived as limitations on human performance. This conflict underlies the sleep "disorders" of jet lag and shiftwork sleep disruption, problems that are not really diseases at all, but instead reflect normal function of circadian timing in the context of extraordinary demands on sleep-wake scheduling. Whatever their proper classification, both jet lag and shiftwork insomnia represent important societal problems deserving of public health and medical attention. Barring a worldwide rejection of air-travel, jet lag will continue to afflict tens of thousands of people annually. The effects of jet lag on human performance, while typically transient, can nonetheless be significant, affecting commerce, government, and even the outcome of professional sports contests. More important, only a global regression to an agrarian economy will eliminate the problem of tens of millions of workers in this country who regularly attempt to work at night and sleep during the day. In contrast to jet lag, shiftwork produces chronic sleep disruption lasting for the duration of shiftwork exposure. For while individual differences in the ability to adjust to a nocturnal work schedule clearly exist, recent studies suggest that few if any night workers regularly experience restful and restorative day sleep equivalent to that considered normal at night. This chronic sleep limitation is associated with significant increases in a number of consequent problems including sleepiness-related accidents, social disruption, and psychiatric disturbances. In addition, chronic exposure to shiftwork has now been shown to be an independent risk factor for the development of both cardiovascular and gastrointestinal diseases. While these epidemiological studies have not identified the specific aspect of shiftwork that is associated with increased risk of these disorders, the chronic limitation and disruption is foremost among plausible factors. The most important aspect of human circadian physiology that limits adaptation to the extreme schedules inherent in shiftwork and jet travel is the primacy of light among entraining signals, or zeitgebers. Exposure to sunlight for night shiftworkers, or for jet travelers at their destination, results in maintenance (or resetting) of the clock to environmental time. This response can be prevented or overridden with extraordinary avoidance of sunlight or with provision of artificial light of sufficient duration and intensity to negate the sunlight signal, an approach shown to be effective in the treatment of shiftwork sleep disruption. Practical issues sharply limit the application of artificial lighting to all shiftwork settings, however, and the role for a pharmacological chronobiotic agent capable of accomplishing the same end is potentially very large (Copinschi et al., 1995; Jamieson et al., 1998). For example, the effects of zolpidem vs. placebo on sleep, daytime alertness, and fatigue in travelers who complain of jet lag was co

Molecular and cellular biology of Alzheimer amyloid.

Alzheimer's Disease (AD), a disorder of unknown etiology, is the most common form of adult-onset dementia and is characterized by severe intellectual deterioration. The definitive diagnosis of AD is made by postmortem examination of the brain, which reveals large quantities of neurofibrillary tangles (NFT) and senile plaques within the parenchyma. The NFT are composed of paired helical filaments associated with several cytoskeletal proteins. The primary protein component of senile plaques is beta/A4 amyloid, a 42-43 amino acid peptide derived from a much larger molecule, the amyloid precursor protein (APP). Vascular beta/A4 amyloidosis is also prevalent in the disease. The mechanism by which beta/A4 amyloid accumulates in the AD brain is unknown. Recent research has demonstrated that the precursor molecule, APP, is a transmembrane protein with a large extracytoplasmic domain, a membrane spanning region that includes the portion that gives rise to beta/A4 amyloid, and a short intracytoplasmic domain. The precursor has multiple forms among which are those that differ by a variable length insert within the extracytoplasmic domain. The insert has sequence homology to the family of Kunitz protease inhibitor proteins. Cellular and animal models have been developed to study the nature of APP processing and the biological and behavioral consequences of beta/A4 amyloidosis. The results of such studies indicate that the normal processing of APP involves enzymatic cleavage of the molecule within the beta/A4 amyloid region, thus preventing the accumulation of beta/A4 in the normal brain. The factors leading to abnormal processing of APP, and consequent beta/A4 amyloid accumulation within the AD brain, have yet to be identified. In cell culture, the biological effects associated with beta/A4 amyloid include neurotrophic and neurotoxic activities, while the peptide has also been shown to have dramatic behavioral effects in animal models.