Effects of hemorrhagic shock and rhabdomyolysis on renal microcirculation, oxygenation and function in a female swine model.

BACKGROUND: Hemorrhagic shock (HS) and rhabdomyolysis (RM) are two important risk factors of acute kidney injury (AKI) after severe trauma, however the effects of the combination of RM and HS on kidney function are unknown. The purpose of this study was to determine the impact of RM and HS on renal function, oxygenation, perfusion and morphology, in a pig model. METHODS: Forty-seven female pigs were divided into 5 groups: sham, RM, HS, HS and moderate RM (RM4/HS), HS and severe RM (RM8/HS). RM was induced by intramuscular injection of Glycerol 50% with a moderate dose (4 ml/kg for RM4/HS group) or a high dose (8 ml/kg for RM and RM8/HS groups). Among animals with HS, after 90 min of hemorrhage, animals were resuscitated with fluid followed by transfusion of the withdrawn blood. Animals were followed for 48 hours. Macro and microcirculatory parameters measurements were performed. RESULTS: RM alone induced a decrease in creatinine clearance at 48 hours (19 (0-41) vs 102 (56-116) ml/min for RM and SHAM respectively; p = 0.0006) without alteration in renal perfusion and oxygenation. HS alone impaired temporarily renal microcirculation, function and oxygenation that were restored with fluid resuscitation. RM4/HS and RM8/HS groups induced greater impairment of renal microcirculation and function than HS alone at the end of blood spoliation that were not improved by fluid resuscitation. Mortality was increased in RM8/HS and RM4/HS groups in the first 48 hours (73% vs 56% vs 9% for RM8/HS, RM4/HS and HS groups respectively). CONCLUSIONS: The combination of HS and RM induced an early deleterious effect on renal microcirculation, function and oxygenation with decreased response to resuscitation and transfusion compared with HS or RM alone.

Renal Microcirculation and Function in a Pig Model of Hemorrhagic Shock Resuscitation with Norepinephrine.

Rationale: Norepinephrine (NE) is commonly used in combination with fluid during resuscitation of hemorrhagic shock, but its impact on kidney microcirculation, oxygenation, and function is still unknown in this setting. Objectives: During hemorrhagic shock resuscitation, does a combination of fluid and NE affect kidney oxygenation tension, kidney microcirculatory perfusion, and 48-hour kidney function, as compared with fluid alone? Methods: Hemorrhagic shock was induced in 24 pigs, and 8 pigs were included as a sham group. Resuscitation of hemorrhagic shock was performed, using a closed-loop device, either by fluid alone (0.9% NaCl; fluid group) or associated with the administration of NE at two doses (moderate dose: mean rate of 0.64 µg ⋅ kg-1 ⋅ min-1; high dose: mean rate of 1.57 µg ⋅ kg-1 ⋅ min-1) to obtain a target systolic arterial pressure of 80 to 90 mm Hg. Resuscitation was followed by transfusion of the withdrawn blood. Measurements and Main Results: The amount of fluid required to reach the target systolic arterial pressure was lower in the NE groups than in the fluid group, with subsequently less hemodilution. NE restored kidney microcirculation, oxygenation, and function in a manner comparable to that achieved with fluid resuscitation alone. There were no histologic differences between animals resuscitated with fluid and those resuscitated with NE. Conclusions: In pigs with hemorrhagic shock, resuscitation with a combination of NE and fluid restored kidney microcirculation and oxygenation, as well as renal function, in a manner comparable to fluid resuscitation alone and without differences between the two NE doses. NE administration led to a fluid volume-sparing effect with subsequently less hemodilution.

Intra-arterial injection of particulate corticosteroids: mechanism of injury.

Mechanism of neurologic complications after epidural spinal injections (ESI) of particulate steroids at the cervical spine include intrathecal injection, epidural hematoma, direct spinal cord injury, and brain stem or cord infarction due to an arterial spasm or inadvertent intra-arterial injection of particulate steroids. At the lumbar spine, there is evidence that a spinal cord infarction secondary to an inadvertent intra-arterial injection of particulate steroids through a transforaminal approach is the leading mechanism.Variations in the arterial supply of the spinal cord help to understand how a lumbar ESI may lead to a spinal cord infarction at the thoracic level. A radiculomedullary artery arising from the lumbar or sacral spine may participate to the supply of the spinal cord. All radicular and radiculomedullary arteries penetrate the spinal canal through the intervertebral foramen. Therefore, its catheterization carries a risk of inadvertent intraarterial injection. An ex vivo animal study has shown that particulate steroids injected in the blood stream produce an immediate and unexpected change of red blood cells into spiculated cells which aggregate and cause arterioles obstruction, while no particulate steroid macroaggregates or vascular spasm were observed. Rare instances of neurologic complications also occurred after ESI performed through a posterior approach. All occurred in previously operated on patients suggesting a pathologic role for the epidural scar.

Cadaveric assessment of a 3D-printed aiming device for implantation of a hinged elbow external fixator.

INTRODUCTION: Proper implantation of a hinged external elbow fixator (HEEF) is demanding since it requires precise alignment between the flexion-extension's and HEEF's axis. In order to optimize this alignment, we have developed a 3D-printed aiming device. The primary goal of the study was to compare the aiming device-based technique with the conventional pin technique. The secondary goal was to determine whether it is possible to share the aiming device with the surgical community. MATERIALS AND METHODS: A HEEF was implanted in cadavers with either the aiming device (n = 6) or the conventional pin technique (n = 6). For both techniques the duration of the procedure, the radiation exposure as well as the offset and angular divergence between the HEEF's and flexion-extension's axis were compared. To achieve the secondary goal, two surgeons used aiming devices 3D-printed from files sent by email in order to implant HEEF on cadaveric specimens (n = 6). RESULTS: Duration of the procedure was not significantly different between both techniques. However, the aiming device allowed for reduction of the number of image intensifier shots (p = 0.005), angular divergence (p = 0.02) and offset between both axes (p = 0.05). The aiming devices have been delivered less than 15 days after ordering, and they have allowed proper implantation of six HEEF. CONCLUSION: The 3D-printed aiming device allowed less irradiant and more accurate implantation of HEEF. It is possible to share it with other surgeons.

Deleterious Effects of Intra-arterial Administration of Particulate Steroids on Microvascular Perfusion in a Mouse Model.

Purpose To determine the in vivo effects of several particulate steroids on microvascular perfusion by using intravital microscopy in a mice model and to investigate the in vitro interactions between these particulate steroids and red blood cells (RBCs). Materials and Methods The study was conducted in agreement with the guidelines of the National Committee of Ethic Reflection on Animal Experimentation. By using intravital microscopy of mouse cremaster muscle, the in vivo effects of several particulate steroids on microvascular perfusion were assessed. Four to five mice were allocated to each of the following treatment groups: saline solution, dexamethasone sodium phosphate, a nonparticulate steroid, and the particulate steroids cortivazol, methylprednisolone, triamcinolone, and prednisolone. By using in vitro blood microcinematography and electron microscopy, the interactions between these steroids and human RBCs were studied. All results were analyzed by using nonparametric tests. Results With prednisolone, methylprednisolone, or triamcinolone, blood flow was rapidly and completely stopped in all the arterioles and venules (median RBC velocity in first-order arterioles, 5 minutes after administration was zero for these three groups) compared with a limited effect in mice treated with saline, dexamethasone, and cortivazol (20.3, 21.3, and 27.5 mm/sec, respectively; P < .003). This effect was associated with a large decrease in the functional capillary density (4.21, 0, and 0 capillaries per millimeter for methylprednisolone, triamcinolone, or prednisolone, respectively, vs 21.0, 21.4, and 19.1 capillaries per millimeter in mice treated with saline, dexamethasone, and cortivazol, respectively; P < .003). This was because of the rapid formation of RBC aggregates. However, no change in microvascular perfusion was associated with administration of cortivazol or dexamethasone. In vitro experiments confirmed the formation of RBC aggregates associated with the transformation of RBCs into spiculated RBCs with the same steroids. Conclusion Several particulate steroids have an immediate and massive effect on microvascular perfusion because of formation of RBC aggregates associated with the transformation of RBCs into spiculated RBCs. (©) RSNA, 2016 Online supplemental material is available for this article.

Effect of norepinephrine on spinal cord blood flow and parenchymal hemorrhage size in acute-phase experimental spinal cord injury.

PURPOSE: In the acute phase of spinal cord injury (SCI), ischemia and parenchymal hemorrhage are believed to worsen the primary lesions induced by mechanical trauma. To minimize ischemia, keeping the mean arterial blood pressure above 85 mmHg for at least 1 week is recommended, and norepinephrine is frequently administered to achieve this goal. However, no experimental study has assessed the effect of norepinephrine on spinal cord blood flow (SCBF) and parenchymal hemorrhage size. We have assessed the effect of norepinephrine on SCBF and parenchymal hemorrhage size within the first hour after experimental SCI. METHODS: A total of 38 animals were included in four groups according to whether SCI was induced and norepinephrine injected. SCI was induced at level Th10 by dropping a 10-g weight from a height of 10 cm. Each experiment lasted 60 min. Norepinephrine was started 15 min after the trauma. SCBF was measured in the ischemic penumbra zone surrounding the trauma epicenter using contrast-enhanced ultrasonography. Hemorrhage size was measured repeatedly on parasagittal B-mode ultrasonography slices. RESULTS: SCI was associated with significant decreases in SCBF (P = 0.0002). Norepinephrine infusion did not significantly modify SCBF. Parenchymal hemorrhage size was significantly greater in the animals given norepinephrine (P = 0.0002). CONCLUSION: In the rat, after a severe SCI at the Th10 level, injection of norepinephrine 15 min after SCI does not modify SCBF and increases the size of the parenchymal hemorrhage.

Rat model of spinal cord injury preserving dura mater integrity and allowing measurements of cerebrospinal fluid pressure and spinal cord blood flow.

PURPOSES: Cerebrospinal fluid (CSF) pressure elevation may worsen spinal cord ischaemia after spinal cord injury (SCI). We developed a rat model to investigate relationships between CSF pressure and spinal cord blood flow (SCBF). METHODS: Male Wistar rats had SCI induced at Th10 (n = 7) or a sham operation (n = 10). SCBF was measured using laser-Doppler and CSF pressure via a sacral catheter. Dural integrity was assessed using subdural methylene-blue injection (n = 5) and myelography (n = 5). RESULTS: The SCI group had significantly lower SCBF (p < 0.0001) and higher CSF pressure (p < 0.0001) values compared to the sham-operated group. Sixty minutes after SCI or sham operation, CSF pressure was 8.6 ± 0.4 mmHg in the SCI group versus 5.5 ± 0.5 mmHg in the sham-operated group. No dural tears were found after SCI. CONCLUSION: Our rat model allows SCBF and CSF pressure measurements after induced SCI. After SCI, CSF pressure significantly increases.

De novo generation in an in vivo rat model and biomechanical characterization of autologous transplants for ligament and tendon reconstruction.

BACKGROUND: Surgical reconstruction of ligaments and tendons is frequently required in clinical practice. The commonly used autografts, allografts, or synthetic transplants present limitations in terms of availability, biocompatibility, cost, and mechanical properties that tissue bioengineering aims to overcome. It classically combines an exogenous extracellular matrix with cells, but this approach remains complex and expensive. Using a rat model, we tested a new bioengineering strategy for the in vivo and de novo generation of autologous grafts without the addition of extracellular matrix or cells, and analyzed their biomechanical and structural properties. METHODS: A silicone perforated tubular implant (PTI) was designed and implanted in the spine of male Wistar rats to generate neo-transplants. The tensile load to failure, stiffness, Young modulus, and ultrastructure of the generated tissue were determined at 6 and 12weeks after surgery. The feasibility of using the transplant that was generated in the spine as an autograft for reconstruction of medial collateral ligaments (MCL) and Achilles tendons was also tested. FINDINGS: Use of the PTI resulted in de novo transplant generation. Their median load to failure and Young modulus increased between 6 and 12weeks (respectively 12N vs 34N and 48MPa vs 178MPa). At 12weeks, the neo-transplants exhibited collagen bundles (mainly type III) parallel to their longitudinal axis and elongated fibroblasts. Six weeks after their transfer to replace the MCL or the Achilles tendon, the transplants were still present, with their ends healed at their insertion point. INTERPRETATION: This animal study is a first step in the design and validation of a new bioengineering strategy to develop autologous transplants for ligament and tendon reconstructions.

Characteristic features of in vivo skin microvascular reactivity in CADASIL.

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is caused by mutations in the Notch3 receptor expressed at the surface of vascular smooth muscle cells. The functional consequences of the disease at the peripheral microcirculation level are incompletely elucidated. In this study, we aimed to assess, in vivo, the endothelium-dependent and independent vasodilation of the skin microvasculature in CADASIL patients. Twenty-three affected subjects were compared with 23 gender and age-matched controls. The brachial artery endothelium-dependent and endothelium-independent vasodilation were assessed after forearm cuff occlusion and nitroglycerin administration. Skin vasoreactivity to transcutaneous administration of acetylcholine and sodium nitroprussiate, and after postocclusive hyperemia were measured by Laser Doppler flowmetry. The maximum changes in the diameter of the brachial artery after the cuff release or after nitroglycerin administration did not differ between patients and controls. With iontopheresis, only the peak value of the dose response was found decreased in normocholesterolemic patients after nitroprussiate administration. The postocclusive test revealed a large increase of the time to peak value and whole duration of the hyperemic response in CADASIL patients. The results of this study show that the skin vasoreactivity is altered in CADASIL. Particularly, the kinetics of reactive hyperemia after cuff occlusion is dramatically changed with a lengthened and delayed response. This characteristic pattern may be related to the specific ultrastructural modifications related to Notch3 gene mutations involving smooth muscle cells in the microvasculature.

Combinatorial therapy with two pro-coagulants and one osmotic agent reduces the extent of the lesion in the acute phase of spinal cord injury in the rat.

BACKGROUND: Spinal cord injury (SCI) is a complex disease that leads to a motor, sensitive, and vegetative impairment. So far, single therapies are ineffective for treating SCI in humans and a multifactorial therapeutic approach may be required. The aim of this work was to assess the effect of a triple therapy (TT) associating two pro-coagulant therapies (tranexamic acid and fibrinogen) with an anti-edema therapy (hypertonic saline solution), on the extent of the lesion 24 h post-injury. METHODS: The design of this study is a randomized controlled study. The setting of this study is an experimental study. Male Wistar rats were assigned to receive saline solution for the control group or one of the treatment, or a combination of two treatments or the three treatments (triple therapy group (TT)). Animals were anesthetized and received a weight-drop SCI induced at the level of the 12th thoracic vertebra (Th12). They were treated by single therapies, double therapies, or TT started 5 min after the SCI. RESULTS: The extent of the lesion was assessed 24 h after injury by spectrophotometry (quantification of parenchymal hemorrhage and blood-spinal cord barrier disruption) and by histology (quantification of spared neuronal tissue). As compared with the control group, the TT significantly reduced parenchymal hemorrhage (p < 0.05) and improved the total amount of intact neural tissue, measured 24 h later (p = 0.003). CONCLUSIONS: Combinatorial therapy associating two pro-coagulants (tranexamic acid and fibrinogen) with an anti-edema therapy (hypertonic saline solution) reduces the extent of the lesion in the acute phase of spinal cord injury in the rat.

Contrast enhanced ultrasound imaging for assessment of spinal cord blood flow in experimental spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 µl) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described "ischemic penumbra zone". This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Effects of adenosine monophosphate used in combination with L-arginine on female rabbit corpus cavernosum tissue.

INTRODUCTION: Sexual dysfunction is significantly more prevalent in women than in men. However, to date, no satisfactory oral treatment is yet available. AIM: The aim of this study was to study the effects of adenosine monophosphate (AMP) alone or its combination with L-Arginine on the relaxation of the female rabbit corpus cavernosum. METHODS: Cylinder strips from the corporal body of the excised clitoris from female New Zealand White rabbits were incubated in Krebs solution. Phenylephrine (PE) precontraction was achieved, then the drugs AMP and L-Arginine were administered either independently or in sequential combinations to the strips under precontracted conditions. MAIN OUTCOME MEASURES: Contraction percentages were compared. RESULTS: When precontraction was induced by PE 8 µM or 20 µM, AMP was shown to induce relaxation up to 25% in a dose-dependent manner. The relaxation induced by L-Arginine reached 15.6% at 5.10(-4) M vs. 16.5% at AMP 5.10(-4) M under the same experimental conditions. Nitric oxide (NO) synthase inhibitor N-nitro-L-arginine strongly inhibited the relaxing effect provoked by AMP, suggesting that the action mechanism of this nucleotide is related to the NO pathway. The combination of L-Arginine at 5.10(-4) M with AMP at different doses ranging from 5.10(-4) M to 10(-3) M significantly amplified the relaxing response up to 40.7% and 58%, respectively. CONCLUSIONS: Our results demonstrate that AMP induces a relaxing effect on the female rabbit corpora. They also show that L-Arginine and AMP can potentiate each other and that a synergistic effect can be obtained by their combined use. Because only slight differences exist between both sexes in response to NO donors and/or nucleotide purines or in their use together, it is very likely that close biochemical mechanisms, although not to the same degree and not quite similar, are involved in the engorgement of the penis and the clitoris of New Zealand White rabbits. Stücker O, Pons C, Neuzillet Y, Laemmel E, and Lebret T. Original research-sexual medicine: Effects of adenosine monophosphate used in combination with L-Arginine on female rabbit corpus cavernosum tissue. Sex Med 2014;2:1-7.

Real-time and spatial quantification using contrast-enhanced ultrasonography of spinal cord perfusion during experimental spinal cord injury.

STUDY DESIGN: Experimental study in male Wistar rats. OBJECTIVE: To quantify temporal and spatial changes simultaneously in spinal cord blood flow and hemorrhage during the first hour after spinal cord injury (SCI), using contrast-enhanced ultrasonography (CEU). SUMMARY OF BACKGROUND DATA: Post-traumatic ischemia and hemorrhage worsen the primary lesions induced by SCI. Previous studies did not simultaneously assess temporal and spatial changes in spinal cord blood flow. METHODS: SCI was induced at Th10 in 12 animals, which were compared with 11 sham-operated controls. Spinal cord blood flow was measured in 7 adjacent regions of interest and in the sum of these 7 regions. Blood flow was quantified using CEU with intravenous microbubble injection. Spinal cord hemorrhage was measured on conventional B-mode sonogram slices. RESULTS: CEU allowed us to measure the temporal and spatial changes in spinal cord blood flow in both groups. In the SCI group, spinal cord blood flow was significantly decreased in the global region of interest (P = 0.0016), at the impact site (epicenter), and in the 4 regions surrounding the epicenter, compared with the sham group. The blood flow decrease was maximum at the epicenter. No statistically significant differences between the sham groups were found for the most rostral and caudal regions of interest. Hemorrhage size increased significantly with time (P < 0.0001), from 30.3 mm(2) (±2) after 5 minutes to 39.6 mm(2) (±2.3) after 60 minutes. CONCLUSION: CEU seems reliable for quantifying temporal and spatial changes in spinal cord blood flow. After SCI, bleeding occurs in the spinal cord parenchyma and increases significantly throughout the first hour.

In vivo reactive oxygen species production induced by ischemia in muscle arterioles of mice: involvement of xanthine oxidase and mitochondria.

Reactive oxygen species (ROS) participate in tissue injury after ischemia-reperfusion. Their implication in leukocyte adherence and increase in permeability at the venular side of the microcirculation have been reported, but very little is known about ROS production in arterioles. The objective of this work was to evaluate, in the arteriole wall in vivo, the temporal changes in superoxide anion production during ischemia and reperfusion and to identify the source of this production. Mouse cremaster muscle was exposed to 1 h of ischemia followed by 30 min of reperfusion, and superoxide anion production was assessed by a fluorescent probe, i.e., intracellular dihydroethidium oxidation. During ischemia, we found a significant increase in dihydroethidium oxidation; however, we observed no additional increase in fluorescence during the subsequent reperfusion. This phenomenon was significantly inhibited by pretreatment with superoxide dismutase. Allopurinol (xanthine oxidase inhibitor) or stigmatellin [Q(o)-site (oriented toward the intermembrane space) inhibitor of mitochondrial complex III] or simultaneous administration of these two inhibitors significantly reduced superoxide production during ischemia to 80%, 88%, and 72%, respectively, of that measured in the untreated ischemia-reperfusion group. By contrast, no significant inhibition was found when NADPH oxidase was inhibited by apocynin or when mitochondrial complex I or complex II was inhibited by rotenone or thenoyltrifluoroacetone. A significant increase in ROS was found with antimycin A [Q(i)-site (located in the inner membrane and facing the mitochondrial matrix) inhibitor of mitochondrial complex III]. We conclude that a significant increase in ROS production occurs during ischemia in the arteriolar wall. This increased production involves both a cytoplasmic source (i.e., xanthine oxidase) and the mitochondrial complex III at the Q(o) site.

Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.

This study investigated the capability of fibered confocal fluorescence microscopy (FCFM) to provide in vivo microvascular observations. FCFM is specifically designed for in vivo in situ observation thanks to a probe composed of a fiber bundle and micro-optics having a diameter as small as 650 microm. In the first part of the study, we compared the main characteristics of FCFM with those of intravital fluorescence microscopy (IFM). A mouse cremaster preparation was used as a common basis to allow for imaging with both modalities. We discussed the feasibility of obtaining quantitative measurements usually provided by IFM in the context of FCFM: morphometry, capillary permeability, functional capillary density, vasoconstriction and dilation effects. In addition, the possibility to visualize fluorescent red blood cells or leukocytes was also evaluated. Phototoxicity issues and limitations of FCFM were also discussed. We showed that FCFM allows observations and measurements usually provided by IFM and that the real-time capability of the system, as well as the flexibility and small diameter of the optical probe enable micro-invasiveness and can extend imaging capabilities for in vivo in situ observations when compared to IFM.