Toward the "Perfect" Shunt: Historical Vignette, Current Efforts, and Future Directions.

As a concept, drainage of excess fluid volume in the cranium has been around for more than 1000 years. Starting with the original decompression-trepanation of Abulcasis to modern programmable shunt systems, to other nonshunt-based treatments such as endoscopic third ventriculostomy and choroid plexus cauterization, we have come far as a field. However, there are still fundamental limitations that shunts have yet to overcome: namely posture-induced over- and underdrainage, the continual need for valve opening pressure especially in pediatric cases, and the failure to reinstall physiologic intracranial pressure dynamics. However, there are groups worldwide, in the clinic, in industry, and in academia, that are trying to ameliorate the current state of the technology within hydrocephalus treatment. This chapter aims to provide a historical overview of hydrocephalus, current challenges in shunt design, what members of the community have done and continue to do to address these challenges, and finally, a definition of the "perfect" shunt is provided and how the authors are working toward it.

Neuroprotection is improved by watertightness of fetal spina bifida repair in the sheep model.

OBJECTIVES: A contributing factor to unsuccessful prenatal spina bifida aperta (SBA) repair via an open approach may be incomplete neurosurgical repair causing persistent in-utero leakage of cerebrospinal fluid (CSF) and exposure of the fetal spinal cord to amniotic fluid. We aimed to investigate the neurostructural and neurofunctional efficacy of watertight prenatal SBA repair in a validated SBA fetal lamb model. METHODS: A well-powered superiority study was conducted in the validated SBA fetal lamb model (n = 7 per group). The outcomes of lambs which underwent watertight or non-watertight multilayer repair through an open approach were compared to those of unrepaired SBA lambs (historical controls) at delivery (term = 145 days). At ∼75 days, fetal lambs underwent standardized induction of lumbar SBA. At ∼100 days, they were assigned to an either watertight or non-watertight layered repair group based on an intraoperative watertightness test using subcutaneous fluorescein injection. At 1-2 days postnatally, as primary outcome, we assessed reversal of hindbrain herniation using magnetic resonance imaging (MRI). Secondary proxies of neuroprotection were: absence of CSF leakage at the repair site; hindlimb motor function based on joint-movement score, locomotor grade and Motor Evoked Potential (MEP); four-score neuroprotection scale, encompassing live birth, complete hindbrain herniation reversal, absence of CSF leakage and joint-movement score ≥ 9/15; and brain and spinal cord histology and immunohistochemistry. As the watertightness test cannot be used clinically due to its invasiveness, we developed a potential surrogate intraoperative three-score skin-repair-quality scale based on visual assessment of the quality of the skin repair (suture inter-run distance ≤ 3 mm, absence of tear and absence of ischemia), with high quality defined by a score ≥ 2/3 and low quality by a score < 2/3, and assessed its relationship with improved outcome. RESULTS: Compared with unrepaired lambs, lambs with watertight repair achieved a high level of neuroprotection (neuroprotection score of 4/4 in 5/7 vs 0/7 lambs) as evidenced by: a significant 100% (vs 14%) reversal of hindbrain herniation on MRI; low CSF leakage (14% vs 100%); better hindlimb motor function, with higher joint-movement score, locomotor grade and MEP area under the curve and peak-to-peak amplitude; higher neuronal density in the hippocampus and corpus callosum; and higher reactive astrogliosis at the SBA lesion epicenter. Conversely, lambs with non-watertight SBA repair did not achieve the same level of neuroprotection (score of 4/4 in 1/7 lambs) compared with unrepaired lambs, with: a non-significant 86% (vs 14%) reversal of hindbrain herniation; high CSF leakage (43% vs 100%); no improvement in motor function; low brain neuron count in both the hippocampus and corpus callosum; and small spinal astroglial cell area at the epicenter. Both watertight layered repair and high (≥ 2/3) intraoperative skin-repair-quality score were associated with improved outcome, but the watertightness test and skin-repair-quality scale could not be used interchangeably due to result discrepancies. CONCLUSIONS: Watertight layered fetal SBA repair is neuroprotective since it improves brain and spinal-cord structure and function in the fetal lamb model. This translational research has important clinical implications. A neurosurgical technique that achieves watertightness should be adopted in all fetal centers to improve neuroprotection. Future clinical studies could assess whether a high skin-repair-quality score (≥ 2/3) correlates with neuroprotection. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Comparison of Morphological and Histological Characteristics of Human and Sheep: Sheep as a Potential Model for Testing Midurethral Slings in vivo.

INTRODUCTION: The sheep was evaluated as a potential model for preclinical evaluation of urethral slings in vivo based on: (1) anatomical measurements of the sheep vagina and (2) histological tissue integration and host response to polypropylene (PP) slings. METHODS: Eight female, multiparous sheep were utilized. Three of 8 animals underwent surgery mimicking human tension-free vaginal tape protocols for midurethral slings and were euthanized at 6 months. The following measurements were obtained: vaginal length, maximum vaginal width with retraction, symphysis pubis length, and distance from the pubic bone to incision. Explanted sling samples from sheep and human were stained with hematoxylin and eosin for host reaction assessment. RESULTS: Geometric measurements were similar between humans and sheep. Sheep vaginal anatomy allowed sling placement similar to procedures in human surgeries, and all sheep recovered without problems. Comparative histology between the sheep and human indicated similar host reaction and collagen deposition around implants, confirming suitability of the sheep model for biomaterial response assessment. CONCLUSION: Sheep vaginal length is comparable to humans. Tissue integration and host response to PP slings showed chronic inflammation with rich collagen deposition around the material in both sheep and human specimens, highlighting the sheep as a potential animal model for preclinical testing of midurethral slings.

Evaluation of microRNA expression in a sheep model for lung fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibroproliferative disorder that has one of the poorest prognoses amongst interstitial lung diseases. Recently, the finding of aberrant expression levels of miRNAs in IPF patients has drawn significant attention to the involvement of these molecules in the pathogenesis of this disease. Clarification of the differential expression of miRNAs in health and disease may identify novel therapeutic strategies that can be employed in the future to combat IPF. This study evaluates the miRNA expression profiles in a sheep model for lung fibrosis and compares them to the miRNA profiles of both IPF patients and the mouse bleomycin model for pulmonary fibrosis. Pathway enrichment analyses were performed on differentially expressed miRNAs to illustrate which biological mechanisms were associated with lung fibrosis. RESULTS: We discovered 49 differentially expressed miRNAs in the sheep fibrosis model, in which 32 miRNAs were significantly down regulated, while 17 miRNAs were significantly upregulated due to bleomycin-induced lung injury. Moreover, the miRNA families miR-29, miR-26, miR-30, let-7, miR-21, miR-19, miR-17 and miR-199 were aberrantly expressed in both sheep and mouse models, with similar differential miRNAs expression observed in IPF cases. Importantly, 18 miRNAs were aberrantly expressed in both the sheep model and IPF patients, but not in mice. CONCLUSION: Together with pathway enrichment analyses, these results show that the sheep model can potentially be used to characterize previously unrecognized biological pathways associated with lung fibrosis.

Effect of Stent Strut Interval on Neointima Formation After Venous Stenting in an Ovine Model.

OBJECTIVE: The impact of stent design on venous patency is not well studied. The purpose of this study was to investigate the effect of stent material burden on endothelial coverage of stented venous segments, which may contribute to vessel healing and patency. METHODS: Segmented self expanding bare nitinol stents (18 × 50 mm) comprising 5 mm long attached metallic rings separated by 2, 5, or 8 mm gaps were implanted in the inferior vena cava (IVC) of 10 sheep. These stents were designed and manufactured for the purposes of this study. At six, 12, and 24 weeks after implantation the animals were euthanised and the stented vessels harvested for histomorphometric analysis. Three sections from the metallic part as well as the gaps between the struts were reviewed for quantification of endothelialisation after six, 12, and 24 weeks. The intimal thickness over and between the stent struts was measured. The endothelialisation score (graded from 1 for complete luminal endothelialisation to 5 for absence of endothelial cells) was determined. RESULTS: All stents were successfully deployed and all 10 sheep survived until the time of harvesting. Macroscopic inspection after 24 weeks showed only partial endothelialisation over stents with 2 mm and 5 mm skipped segments, whereas the stents with 8 mm skipped segments were totally incorporated into the vein wall. After 24 weeks, the mean (SD) neointimal thicknesses over stent struts with 2 mm, 5 mm, and 8 mm skipped segments were 254.0 (51.6), 182.2 (98.1), and 194.6 (101.1) µm, respectively. Comparison of endothelialisation scores of stents over time showed statistically significantly better endothelialisation over stents with 8 mm gaps after 12 and 24 weeks. CONCLUSION: Stent designs providing structural support to veins with larger gaps between the scaffold material appear to lead to faster and more complete endothelialisation as well as a thinner intimal layer.

Local tissue effects and peri-implant bone healing induced by implant surface treatment: an in vivo study in the sheep.

OBJECTIVE: The aim of this study was to assess, through biological analysis, the local effects and osseointegration of dental implants incorporating surface micro/nanofeatures compared with implants of identical design without surface treatment. BACKGROUND: Known to impact bone cell behavior, surface chemical and topography modifications target improved osseointegration and long-term success of dental implants. Very few studies assess the performance of implants presenting both micro- and nanofeatures in vivo on the animal models used in preclinical studies for medical device certification. METHODS: Implant surfaces were characterized in terms of topography and surface chemical composition. After 4 weeks and 13 weeks of implantation in sheep femoral condyles, forty implants were evaluated through micro-computed tomography, histopathologic, and histomorphometric analyses. RESULTS: No local adverse effects were observed around implants. Histomorphometric analyses showed significantly higher bone-to-implant contact in the coronal region of the surface-treated implant at week 4 and week 13, respectively, was 79.3 ± 11.2% and 86.4 ± 6.7%, compared with the untreated implants (68.3 ± 8.8% and 74.8 ± 13%). Micro-computed tomography analyses revealed that healing patterns differed between coronal and apical regions, with higher coronal bone-to-implant contact at week 13. Histopathologic results showed, at week 13, bone healing around the surface-treated implant with undistinguishable defect margins, while the untreated implant still presented bone condensation and traces of the initial drill defect. CONCLUSION: Our results suggest that the surface-treated implant not only shows no deleterious effects on local tissues but also promotes faster bone healing around the implant.

Minimally Invasive Precise Application of Bioadhesives to Prevent IPPROM on a Pregnant Sheep Model.

INTRODUCTION: Iatrogenic preterm premature rupture of the membrane remains the Achille's heel of fetoscopy. The aim of this study was to show in vivo feasibility of fetal membrane (FM) defect sealing by the application of tissue glues with umbrella-shaped receptors. METHODS: First, we adapted our previously described ex vivo strategy and evaluated the adhesion strength of different tissue glues, Histoacryl® and Glubran2®, by bonding polytetrafluoroethylene or silicone encapsulated nitinol glue receptor to human FM. Then, we exposed pregnant sheep uterus through a laparotomy and placed a 10-French trocar into the amniotic cavity through which the umbrella-shaped glue receptor (n = 9) was inserted and fixated onto the FM with the tissue glues (n = 8). The tightness of the sealed defects was assessed 4 h post-surgery. RESULTS: Both tissue glues tested resulted in adhesion of the glue receptors to the FM ex vivo. In vivo, all glue receptors opened in the amniotic cavity (n = 9) and all successfully placed glue receptors sealed the FM defect (n = 8). Four hours post-surgery, 2 treatment sites showed minimal leakage whereas the negative control without glue (n = 1) showed substantial leakage. DISCUSSION: This in vivo study confirms that fetoscopically induced FM defects can be sealed by the application of tissue adhesives.

Establishment of a Sheep Model for Hind Limb Peripheral Nerve Injury: Common Peroneal Nerve.

Thousands of people worldwide suffer from peripheral nerve injuries and must deal daily with the resulting physiological and functional deficits. Recent advances in this field are still insufficient to guarantee adequate outcomes, and the development of new and compelling therapeutic options require the use of valid preclinical models that effectively replicate the characteristics and challenges associated with these injuries in humans. In this study, we established a sheep model for common peroneal nerve injuries that can be applied in preclinical research with the advantages associated with the use of large animal models. The anatomy of the common peroneal nerve and topographically related nerves, the functional consequences of its injury and a neurological examination directed at this nerve have been described. Furthermore, the surgical protocol for accessing the common peroneal nerve, the induction of different types of nerve damage and the application of possible therapeutic options were described. Finally, a preliminary morphological and stereological study was carried out to establish control values for the healthy common peroneal nerves regarding this animal model and to identify preliminary differences between therapeutic methods. This study allowed to define the described lateral incision as the best to access the common peroneal nerve, besides establishing 12 and 24 weeks as the minimum periods to study lesions of axonotmesis and neurotmesis, respectively, in this specie. The post-mortem evaluation of the harvested nerves allowed to register stereological values for healthy common peroneal nerves to be used as controls in future studies, and to establish preliminary values associated with the therapeutic performance of the different applied options, although limited by a small sample size, thus requiring further validation studies. Finally, this study demonstrated that the sheep is a valid model of peripheral nerve injury to be used in pre-clinical and translational works and to evaluate the efficacy and safety of nerve injury therapeutic options before its clinical application in humans and veterinary patients.

The in vivo biocompatibility of titanized polypropylene lightweight mesh is superior to that of conventional polypropylene mesh.

OBJECTIVE: To evaluate the histological response to and changes in the biomechanical properties of titanized polypropylene lightweight mesh and conventional polypropylene mesh at 1 and 12 weeks following implantation in the sheep vagina. METHODS: We compared a titanized polypropylene lightweight mesh (TiLOOP Mesh) to a conventional polypropylene mesh (Gynemesh PS) in a sheep vagina model. Explants were harvested after 1 and 12 weeks (n = 6/mesh type/time point) for histological observation. After 12 weeks, mesh-tissue complex specimens were biomechanically assessed by a uniaxial tension system. RESULTS: One week after implantation, there was no significant difference in the inflammatory response between the two groups. Twelve weeks after implantation, the TiLOOP light mesh elicited a lower inflammatory response than was observed for the Gynemesh PS (1.44 ± 0.61 vs 2.05 ± 0.80, P = .015). Twelve weeks after implantation, the collagen I/III ratio was lower in the TiLOOP light mesh group than in the Gynemesh PS group (9.41 ± 5.06 vs 15.21 ± 8.21, P = .019). The messenger RNA expression levels of the inflammatory factors interleukin 10 and tumor necrosis factor α were lower in the TiLOOP Mesh group than in the Gynemesh PS group at both 1 and 12 weeks (P < .05). There were no significant differences in any of the evaluated biomechanical characteristics between the two meshes (P > .05). CONCLUSION: Although the titanized polypropylene lightweight mesh induces slightly less tissue reactivity and has better in vivo biocompatibility, further studies should be conducted including the complications and the success rate of pelvic organ prolapse in patients before recommending it in pelvic floor reconstruction.

Experimental study of a new original mesh developed for pelvic floor reconstructive surgery.

INTRODUCTION AND HYPOTHESIS: Most synthetic meshes used in transvaginal surgery are made of polypropylene, which has a stable performance, but does not easily degrade in vivo. However, mesh-related complications are difficult to address and have raised serious concerns. A new biomaterial mesh with good tissue integration and few mesh-related complications is needed. To evaluate the effect of a new bacterial cellulose (BC) mesh on pelvic floor reconstruction following implantation in the vagina of sheep after 1 and 12 weeks. METHODS: The meshes were implanted in the submucosa of the posterior vagina wall of sheep. At 1 and 12 weeks after surgery, mesh-tissue complex (MTC) specimens were harvested for histological studies and biomechanical evaluation. At 12 weeks after surgery, MTC specimens were biomechanically assessed by a uniaxial tension "pulley system". RESULTS: The BC mesh elicited a higher inflammatory response than Gynemesh™PS at both 1 and 12 weeks after implantation. Twelve weeks after implantation, the BC mesh resulted in less fibrosis than Gynemesh™PS. Compared with the Gynemesh™PS group, the BC mesh group had increased mRNA expression of MMP-1, MMP-2, and MMP-9 (P < 0.05), but decreased expression of the anti-inflammatory factor IL-4 (P < 0.05). Twelve weeks after implantation, the ultimate load and maximum elongation percentage of the BC mesh were significantly lower than those of Gynemesh™PS. CONCLUSIONS: The BC mesh could not be a promising biomaterial for pelvic floor reconstructive surgery unless the production process and parameters were improved.

Comparative effectiveness of PEEK rods versus titanium alloy rods in cervical fusion in a new sheep model.

BACKGROUND: Pedicle screw and rod instrumentation based on titanium can produce satisfying strength and stiffness for spinal fusion. However, excessive stiffness produced by titanium rods may cause stress shielding. Thus, polyetheretherketone (PEEK) rods with a low modulus of elasticity were introduced as substitutes for titanium rods. The purpose of this paper is to compare the effectiveness of PEEK rods versus titanium alloy rods in anterior spinal fusion with a new sheep model. METHODS: Sheep models of anterior-posterior cervical fusion were innovatively adopted in our study. Twenty-four sheep were randomly divided into a control group and a treatment group that received anterior-posterior cervical fixation with titanium rods or PEEK rods, respectively. Then, surgical segments were harvested and assessed by X-ray, micro-CT and histological examination to evaluate the efficiency of bone fusion. RESULTS: No complications related to fixation were found during the research process. The results of the X-ray showed a stronger spinal fusion in the PEEK rod groups than in the titanium rod group at 12 weeks postoperatively, and both groups underwent bone fusion at 24 weeks postoperatively. The results of micro-CT showed that fixation with PEEK rods achieved better bone ingrowth at an early postoperative stage (12 weeks) compared to fixation with titanium rods (bone volume fraction (BVF): 20.26 ± 4.36% vs 14.48 ± 3.49%, p < 0.05). The same trend was detected in the histological analysis, where the mineralized bone fraction in the experiment group (21.01 ± 3.48%) was significantly higher than that in the control group (16.73 ± 2.95%). In addition, better osseointegration was found in the experiment group at the early postoperative stage at 12 weeks (bone apposition (BA): 16.22 ± 3.24% vs 11.67 ± 3.63%, p < 0.05). However, there were no significant differences at 24 weeks postoperatively. CONCLUSION: PEEK rods can be used safely in a sheep model of anterior-posterior cervical fixation. Compared to traditional titanium rods, earlier and more evident bone fusion was found in the PEEK rods group. These slides can be retrieved under Electronic Supplementary Material.

Altered Cerebral Blood Flow and Potential Neuroprotective Effect of Human Relaxin-2 (Serelaxin) During Hypoxia or Severe Hypovolemia in a Sheep Model.

Specific neuroprotective strategies to minimize cerebral damage caused by severe hypoxia or hypovolemia are lacking. Based on previous studies showing that relaxin-2/serelaxin increases cortical cerebral blood flow, we postulated that serelaxin might provide a neuroprotective effect. Therefore, we tested serelaxin in two emergency models: hypoxia was induced via inhalation of 5% oxygen and 95% nitrogen for 12 min; thereafter, the animals were reoxygenated. Hypovolemia was induced and maintained for 20 min by removal of 50% of the total blood volume; thereafter, the animals were retransfused. In each damage model, the serelaxin group received an intravenous injection of 30 µg/kg of serelaxin in saline, while control animals received saline only. Blood gases, shock index values, heart frequency, blood pressure, and renal blood flow showed almost no significant differences between control and treatment groups in both settings. However, serelaxin significantly blunted the increase of lactate during hypovolemia. Serelaxin treatment resulted in significantly elevated cortical cerebral blood flow (CBF) in both damage models, compared with the respective control groups. Measurements of the neuroproteins S100B and neuron-specific enolase in cerebrospinal fluid revealed a neuroprotective effect of serelaxin treatment in both hypoxic and hypovolemic animals, whereas in control animals, neuroproteins increased during the experiment. Western blotting showed the expression of relaxin receptors and indicated region-specific differences in relaxin receptor-mediated signaling in cortical and subcortical brain arterioles, respectively. Our findings support the hypothesis that serelaxin is a potential neuroprotectant during hypoxia and hypovolemia. Due to its preferential improvement of cortical CBF, serelaxin might reduce cognitive impairments associated with these emergencies.

Femoral osteochondroplasty can be performed effectively without the risk of avascular necrosis or femoral neck fractures in an experimental ovine FAI model.

OBJECTIVE: The experimental induction of cam-type femoroacetabular impingement (FAI) in sheep is established. To tap the full potential of this ovine model, one should be able to perform a femoral osteochondroplasty safely. This study was based on previous cadaver experiments on the blood supply to the ovine femoral head and on the biomechanical strength of the proximal femur following offset creation. We hypothesized that offset creation in this ovine FAI model does not lead to (1) avascular necrosis (AVN) of the ovine femoral head or (2) iatrogenic femoral neck fractures and (3) can be performed effectively. DESIGN: In this experimental, controlled, prospective study nine sheep underwent unilateral FAI induction through an intertrochanteric, varus osteotomy. Seventy days following FAI induction, femoral osteochondroplasty was performed. Sheep were sacrificed after another 140 days. Radiographs, computed tomography (CT) scans and MRI were acquired. Histologic samples were stained with hematoxylin-eosin. (1) The multimodal Association Research Circulation Osseous (ARCO) classification was used for assessment of AVN. (2) Femoral neck fractures were assessed with the multimodal imaging approach. (3) Pre- and postoperative (=after sacrifice) alpha angles and femoral neck diameters were compared. RESULTS: (1) No signs for AVN according to the ARCO classification or (2) for femoral neck fractures were detected. (3) Mean alpha angles and femoral neck diameters decreased significantly (p < 0.001) superiorly by at least 30° respectively 4 mm after the offset creation. CONCLUSIONS: Femoral osteochondroplasty can be performed effectively and without the risk of AVN or femoral neck fractures in this ovine FAI model.

Feasibility of detecting myocardial infarction in the sheep fetus using late gadolinium enhancement CMR imaging.

BACKGROUND: Late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) imaging has enabled the accurate assessment of myocardial infarction (MI). However, LGE CMR has not been performed successfully in the fetus, where it could be useful for animal studies of interventions to promote cardiac regeneration. We believe that LGE imaging could allow us to document the presence, extent and effect of MI in utero and would thereby expand our capacity for conducting fetal sheep MI research. We therefore aimed to investigate the feasibility of using LGE to detect MI in sheep fetuses. METHODS: Six sheep fetuses underwent a thoracotomy and ligation of a left anterior descending (LAD) coronary artery branch; while two fetuses underwent a sham surgery. LGE CMR was performed in a subset of fetuses immediately after the surgery and three days later. Early gadolinium enhancement (EGE) CMR was also performed in a subset of fetuses on both days. Cine imaging of the heart was performed to measure ventricular function. RESULTS: The imaging performed immediately after LAD ligation revealed no evidence of infarct on LGE (n=3). Two of four infarcted fetuses (50%) showed hypoenhancement at the infarct site on the EGE images. Three days after the ligation, LGE images revealed a clear, hyper-enhanced infarct zone in four of the five infarcted fetuses (80%). No hyper-enhanced infarct zone was seen on the one sham fetus that underwent LGE CMR. No hypoenhancement could be seen in the EGE images in either the sham (n=1) or the infarcted fetus (n=1). No regional wall motion abnormalities were apparent in two of the five infarcted fetuses. CONCLUSION: LGE CMR detected the MI three days after LAD ligation, but not immediately after. Using available methods, EGE imaging was less useful for detecting deficits in perfusion. Our study provides evidence for the ability of a non-invasive tool to monitor the progression of cardiac repair and damage in fetuses with MI. However, further investigation into the optimal timing of LGE and EGE scans and improvement of the sequences should be pursued with the aim of expanding our capacity to monitor cardiac regeneration after MI in fetal sheep.

Glycosphingolipid analysis in a naturally occurring ovine model of acute neuronopathic Gaucher disease.

Gaucher disease arises from mutations in the ß-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the ß-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in ß-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process.

Increase of cortical cerebral blood flow and further cerebral microcirculatory effects of Serelaxin in a sheep model.

Serelaxin, recombinant human relaxin-2, modulates endothelial vasodilatory functionality and is under evaluation for treatment of acute heart failure. Little is known about acute effects on cerebral perfusion. We tested the hypothesis that Serelaxin might also have effects on the cerebral microcirculation in a sheep model, which resembles human brain structure quite well. We used laser Doppler flowmetry and sidestream dark-field (SDF) imaging techniques, which are reliable tools to continuously assess dynamic changes in cerebral perfusion. Laser Doppler flowmetry shows that bolus injection of 30 µg Serelaxin/kg body wt induces an increase (P = 0.006) to roughly 150% of cortical cerebral blood flow (CBF), whereas subcortical CBF remains unchanged (P = 0.688). The effects on area-dependent CBF were significantly different after the bolus injection (P = 0.042). Effects on cortical CBF were further confirmed by SDF imaging. The bolus injection of Serelaxin increased total vessel density to 127% (P = 0.00046), perfused vessel density to 145% (P = 0.024), and perfused capillary density to 153% (P = 0.024). Western blotting confirmed the expression of relaxin receptors RXFP1 and truncated RXFP2-variants in the respective brain regions, suggesting a possible contribution of RXFP1 on the effects of Serelaxin. In conclusion, the injection of a high dose of Serelaxin exerts quick effects on the cerebral microcirculation. Therefore, Serelaxin might be suitable to improve cortical microcirculation and exert neuroprotective effects in clinically relevant scenarios that involve cortical hypoperfusion. These findings need to be confirmed in relevant experimental settings involving cerebral cortical hypoperfusion and can possibly be translated into clinical practice.

Addition of 20-kDa PEG to Insulin Lispro Alters Absorption and Decreases Clearance in Animals.

PURPOSE: Determine the pharmacokinetics of insulin peglispro (BIL) in 5/6-nephrectomized rats and study the absorption in lymph duct cannulated (LDC) sheep. METHODS: BIL is insulin lispro modified with 20-kDa linear PEG at lysine B28 increasing the hydrodynamic size to 4-fold larger than insulin lispro. Pharmacokinetics of BIL and insulin lispro after IV administration were compared in 5/6-nephrectomized and sham rats. BIL was administered IV or SC into the interdigital space of the hind leg, and peripheral lymph and/or serum samples were collected from both LDC and non-LDC sheep to determine pharmacokinetics and absorption route of BIL. RESULTS: The clearance of BIL was similar in 5/6-nephrectomized and sham rats, while the clearance of insulin lispro was 3.3-fold slower in 5/6-nephrectomized rats than in the sham rats. In non-LDC sheep, the terminal half-life after SC was about twice as long vs IV suggesting flip-flop pharmacokinetics. In LDC sheep, bioavailability decreased to <2%; most of the dose was absorbed via the lymphatic system, with 88% ± 19% of the dose collected in the lymph after SC administration. CONCLUSION: This work demonstrates that increasing the hydrodynamic size of insulin lispro through PEGylation can impact both absorption and clearance to prolong drug action.

Intrabody application of eptotermin alpha enhances bone formation in osteoporotic fractures of the lumbar spine; however, fails to increase biomechanical stability - results of an experimental sheep model.

This study analyses the effect of eptotermin α application into fractured vertebrae. It is hypothesized that eptotermin α is capable to enhance bony healing of the osteoporotic spine. In 10 Merino sheep osteoporosis induction was performed by ovariectomy, corticosteroid therapy and calcium/phosphorus/vitamin D-deficient diet; followed by standardized creation of lumbar vertebral compression fractures (VCFs) type A3.1 and consecutive fracture reduction/fixation using expandable mesh cages. Randomly, intravertebral eptotermin α (G1) or no augmentation was added (G2). Macroscopic, micro-CT, and biomechanical evaluation assessed bony consolidation two months postoperatively: Micro-CT data revealed bony consolidation for all cases with significant increased callus development for G2 (60%) and BV/TV (bone volume/total volume 73.45%, osteoporotic vertebrae 35.76%). Neither group showed improved biomechanical stability. Eptotermin α enhanced mineralisation in VCFs in an experimental setup with use of cementless augmentation via an expandable cage. However, higher bone mineral density did not lead to superior biomechanical properties.

Does prolonged post-mortem cold ischemic harvesting time influence cryopreserved pulmonary homograft tissue integrity?

This study investigated cryopreserved pulmonary homograft (CPA) structural integrity after prolonged cold ischemic harvesting times in a juvenile sheep model. Three groups with different post-mortem cold ischemic harvesting times were studied, i.e. Group 1 (24 h, n = 10); group 2 (48 h, n = 10); group 3 (72 h, n = 10). In each group, 5 CPAs were studied in vitro after cryopreservation and thawing. The other 5 CPAs were implanted in juvenile sheep for a minimum of 180 days. Serology samples were obtained and echocardiography was performed before euthanasia. Hematoxylin and eosin (H&E), scanning electron microscopy (SEM), von Kossa, Picrosirius red, α-actin, immunohistochemistry [von Willebrand factor (vWF), CD4, CD31 and CD34] and calcium content analyses were performed on explanted CPAs. The in vitro and in vivo studies failed to demonstrate any change in tensile strength, Young's Modulus and thermal denaturation (Td) results between the groups. SEM demonstrated a reduction in endothelial cells (50 % at 24 h, 60.9 % at 48 h and 40.9 % at 72 h), but H&E could not demonstrate autolysis in any CPA in vitro. All cultures were negative. In the explanted groups, IgE, IgM and IgG results were inconclusive. Echocardiography demonstrated normal valve function in all groups. H&E and Picrosirius red staining confirmed tissue integrity. vWF, CD31 and CD34 staining confirmed a monolayer of endothelial cells in all explanted valves. Calcium content of explanted CPA leaflets was similar. This experimental study supports the concept of prolonging the cold ischemic harvesting time of cryopreserved homografts to reduce homograft shortage.

Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model.

BACKGROUND AIMS: Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. METHODS: We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4(+) fraction of CD34(+) HSCs, we could improve engraftment. Human cord blood-derived CD34(+) cells and human bone marrow-derived MSCs were used for these studies. RESULTS: When MSCs were transplanted 1 week before CD34(+) cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34(+) cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4(+) cells in the CD34(+) population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. CONCLUSIONS: Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.