Klaus-Dieter Schaser

Prolonged Superficial Local Cryotherapy Attenuates Microcirculatory Impairment, Regional Inflammation, and Muscle Necrosis after Closed Soft Tissue Injury in Rats

Background Closed soft tissue injury induces progressive microvascular dysfunction and regional inflammation. The authors tested the hypothesis that adverse trauma-induced effects can be reduced by local cooling. While superficial cooling reduces swelling, pain, and cellular oxygen demand, the effects of cryotherapy on posttraumatic microcirculation are incompletely understood. Study Design Controlled laboratory study. Methods After a standardized closed soft tissue injury to the left tibial compartment, male rats were randomly subjected to percutaneous perfusion for 6 hours with 0.9% NaCL (controls; room temperature) or cold NaCL (cryotherapy; 8°C) (n = 7 per group). Uninjured rats served as shams (n = 7). Microcirculatory changes and leukocyte adherence were determined by intravital microscopy. Intramuscular pressure was measured, and invasion of granulocytes and macrophages was assessed by immunohistochemistry. Edema and tissue damage was quantified by gravimetry and decreased desmin staining. Results Closed soft tissue injury significantly decreased functional capillary density (240 ± 12 cm-1); increased microvascular permeability (0.75 ± 0.03), endothelial leukocyte adherence (995 ± 77/cm2), granulocyte (182.0 ± 25.5/mm2) and macrophage infiltration, edema formation, and myonecrosis (ratio: 2.95 ± 0.45) within the left extensor digitorum longus muscle. Cryotherapy for 6 hours significantly restored diminished functional capillary density (393 ± 35), markedly decreased elevated intramuscular pressure, reduced the number of adhering (462 ± 188/cm2) and invading granulocytes (119 ± 28), and attenuated tissue damage (ratio: 1.7 ± 0.17). Conclusion The hypothesis that prolonged cooling reduces posttraumatic microvascular dysfunction, inflammation, and structural impairment was confirmed. Clinical Relevance These results may have therapeutic implications as cryotherapy after closed soft tissue injury is a valuable therapeutic approach to improve nutritive perfusion and attenuate leukocyte-mediated tissue destruction. The risk for evolving compartment syndrome may be reduced, thereby preventing further irreversible aggravation.

Locked internal fixator: sensitivity of screw/plate stability to the correct insertion angle of the screw.

Objective: Internal fixators with angular stability have been developed to provide high stability without compression of the plate on to the bone. Angular and axial stability of a plate–screw construct can be achieved using a conically threaded screw head undersurface and a corresponding conically threaded plate hole. Furthermore, the insertion angle of the screw must correspond precisely to the axis of the screw hole. This is not always achieved in clinical practice and may result in screw loosening. The objective of this study was to examine the relationship between the stability of the locked screw-plate on the insertion angle of the screw. Methods: Locking screws were inserted in an isolated (Point Contact Fixator, PC-Fix) or combined (Locking Compression Plate, LCP 4.5) locking hole with the use of an aiming device. The optimal insertion angle for these plates is perpendicular to the plate surface. The screws were inserted with an axis deviation of 0° (optimal condition), 5°, and 10° respective to the optimal angle (variance ± 1°). The samples were tested under shear or axial (push out) loading conditions until failure occurred. An Instron materials testing machine was used. Results: Locking screws inserted in the isolated locking hole (PC-Fix) showed a significant decrease of failure load if inserted at 5° and 10° angle. Using an optimal insertion angle (0°), failure load was 1480 ± 390 N, with 5° axis deviation 780 ± 160 N, P = 0.0001, and with 10° axis deviation 550 ± 110 N, P = 0.0001. Screws inserted in the combined locking hole (LCP) also showed a significant decrease of push-out force of 77% (4960 ± 1000 N versus 1120 ± 400 N) with 10° axis deviation. Compared to optimal insertion angle (0°), bending load to failure did decrease up to 69% (1240 ± 210 N vs. 390 ± 100 N) with 10° axis deviation. Conclusion: A locking head screw exhibits high stability with a moderate axis deviation in the angle of insertion of up to 5°. However, there is a significant decrease in stability with increasing axis deviation (>5°). An aiming device is recommended to provide optimal fixation with angular stability.

Effects of early and late intravenous norepinephrine infusion on cerebral perfusion, microcirculation, brain-tissue oxygenation, and edema formation in brain-injured rats.

ObjectivesReduction of cerebral perfusion during the early phase after traumatic brain injury is followed by a later phase of normal to increased perfusion. Thus, pharmacologically elevating mean arterial blood pressure with the aim of improving cerebral perfusion may exert different time-dependent effects on cortical perfusion, microcirculation, tissue oxygenation and brain edema formation after traumatic brain injury. DesignRandomized, placebo-controlled trial. SettingExperimental laboratory at a university hospital. SubjectsA total of 37 male Sprague-Dawley rats subjected to a focal cortical contusion. InterventionsAt 4 or 24 hrs after focal traumatic brain injury, mean arterial blood pressure was increased to 120 mm Hg for 90 mins by infusing norepinephrine. In rats receiving physiologic saline, mean arterial blood pressure remained unchanged. In the first series, pericontusional cortical perfusion was measured using the laser Doppler flowmetry scanning technique before injury and before, during, and after the infusion period. In a second series, intracranial and cerebral perfusion pressure and intraparenchymal perfusion and tissue oxygen measured within the contused and pericontusional cortex were recorded continuously before, during, and after norepinephrine infusion. Changes in cortical microcirculation were investigated by orthogonal polarization spectral imaging. At the end of each experiment, hemispheric swelling and water content were determined gravimetrically. Measurements and Main ResultsAt 4 and 24 hrs after traumatic brain injury, intravenous norepinephrine significantly increased pericontusional cortical perfusion, which was also reflected by an increase in diameters and flow velocities of pericontusional arterioles and venules. Cerebral perfusion pressure and intraparenchymal perfusion and tissue oxygen were significantly increased during norepinephrine infusion at 4 and 24 hrs. Hemispheric swelling and water content showed no difference between the groups. ConclusionsAfter cortical impact injury, early and late intravenous norepinephrine infusion pressure-dependently increased cerebral perfusion and tissue oxygenation without aggravating or reducing brain edema formation. Future studies are warranted to determine long-term changes of short and prolonged norepinephrine-induced increases in mean arterial blood pressure and cerebral perfusion pressure.

Exome sequencing of osteosarcoma reveals mutation signatures reminiscent of BRCA deficiency

Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.

Temporal profile of cortical perfusion and microcirculation after controlled cortical impact injury in rats

Impaired cerebral perfusion contributes to evolving posttraumatic tissue damage. Spontaneous reversibility of reduced perfusion within the first days after injury could make a persisting impact on secondary tissue damage less likely and needs to be considered for possible therapeutic approaches. The present study was designed to characterize the temporal profile and impact of trauma severity on cortical perfusion and microcirculation during the first 48 h after controlled cortical impact injury (CCI). In 10 rats, pericontusional cortical perfusion and microcirculation using laser Doppler flowmetry (LDF) and orthogonal polarization spectral (OPS) imaging were assessed before, and at 4, 24, and 48 h after CCI. Influence of trauma severity was studied by varying the penetration depth of the impactor rod (0.5 vs. 1 mm), thereby inducing a less and a more severe contusion. Mean arterial blood pressure (MABP), arterial blood gases, and blood glucose were monitored. With unchanged MABP and paCO2, cortical perfusion and microcirculation were significantly impaired during the first 48 h following CCI. Hypoperfusion observed at 4 h related to vasoconstriction and microcirculatory stasis preceded a long-lasting phase of hyperperfusion at 24 and 48 h reflected by vasodilation and increased flow velocity in arterioles and venules. Hyperperfusion was mostly pronounced in rats with a less severe contusion. Following CCI, trauma severity markedly influences changes in pericontusional cortical perfusion and microcirculation. Overall, pericontusional cortical hypoperfusion observed within the early phase preceded a long lasting phase of hyperperfusion up to 48 h after CCI.

Biaxial cell stimulation: A mechanical validation

To analyse mechanotransduction resulting from tensile loading under defined conditions, various devices for in vitro cell stimulation have been developed. This work aimed to determine the strain distribution on the membrane of a commercially available device and its consistency with rising cycle numbers, as well as the amount of strain transferred to adherent cells. The strains and their behaviour within the stimulation device were determined using digital image correlation (DIC). The strain transferred to cells was measured on eGFP-transfected bone marrow-derived cells imaged with a fluorescence microscope. The analysis was performed by determining the coordinates of prominent positions on the cells, calculating vectors between the coordinates and their length changes with increasing applied tensile strain. The stimulation device was found to apply homogeneous (mean of standard deviations approx. 2% of mean strain) and reproducible strains in the central well area. However, on average, only half of the applied strain was transferred to the bone marrow-derived cells. Furthermore, the strain measured within the device increased significantly with an increasing number of cycles while the membranes Youngs modulus decreased, indicating permanent changes in the material during extended use. Thus, strain magnitudes do not match the system readout and results require careful interpretation, especially at high cycle numbers.

Expression of survivin detected by immunohistochemistry in the cytoplasm and in the nucleus is associated with prognosis of leiomyosarcoma and synovial sarcoma patients.

BackgroundSurvivin, a member of the inhibitor of apoptosis-protein family suppresses apoptosis and regulates cell division. It is strongly overexpressed in the vast majority of cancers. We were interested if survivin detected by immunohistochemistry has prognostic relevance especially for patients of the two soft tissue sarcoma entities leiomyosarcoma and synovial sarcoma.MethodsTumors of leiomyosarcoma (n = 24) and synovial sarcoma patients (n = 26) were investigated for their expression of survivin by immunohistochemistry. Survivin expression was assessed in the cytoplasm and the nucleus of tumor cells using an immunoreactive scoring system (IRS).ResultsWe detected a survivin expression (IRS > 2) in the cytoplasm of 20 leiomyosarcomas and 22 synovial sarcomas and in the nucleus of 12 leiomyosarcomas and 9 synovial sarcomas, respectively. There was no significant difference between leiomyosarcoma and synovial sarcoma samples in their cytoplasmic or nuclear expression of survivin. Next, all sarcoma patients were separated in four groups according to their survivin expression in the cytoplasm and in the nucleus: group 1: negative (IRS 0 to 2); group 2: weak (IRS 3 to 4); group 3: moderate (IRS 6 to 8); group 4: strong (IRS 9 to 12). In a multivariate Coxs regression hazard analysis survivin expression detected in the cytoplasm or in the nucleus was significantly associated with overall survival of patients in group 3 (RR = 5.7; P = 0.004 and RR = 5.7; P = 0.022, respectively) compared to group 2 (reference). Patients whose tumors showed both a moderate/strong expression of survivin in the cytoplasm and a moderate expression of survivin in the nucleus (in both compartments IRS ≥ 6) possessed a 24.8-fold increased risk of tumor-related death (P = 0.003) compared to patients with a weak expression of survivin both in the cytoplasm and in the nucleus.ConclusionSurvivin protein expression in the cytoplasma and in the nucleus detected by immunohistochemistry is significantly associated with prognosis of leiomyosarcoma and synovial sarcoma patients.

Effects of LY379268, a Selective Group II Metabotropic Glutamate Receptor Agonist on EEG Activity, Cortical Perfusion, Tissue Damage, and Cortical Glutamate, Glucose, and Lactate Levels in Brain-Injured Rats

Activating presynaptic group II metabotropic glutamate (mGlu II) receptors reduces synaptic glutamate release. Attenuating glutamatergic transmission without blocking ionotropic glutamate receptors, thus avoiding unfavorable psychomimetic side effects, makes mGlu II receptor agonists a promising target in treating brain-injured patients. Neuroprotective effects of LY379268 were investigated in rats following controlled cortical impact injury (CCI). At 30 min after CCI, rats received a single intraperitoneal injection of LY379268 (10 mg/kg/body weight) or NaCl. Changes in EEG activity and pericontusional cortical perfusion were determined before trauma, at 4, 24, and 48 h, and 7 days after CCI. Brain edema and contusion volume were determined at 24 h and 7 days after CCI, respectively. Before brain removal pericontusional cortical glutamate, glucose, and lactate were measured via microdialysis. During the early period following CCI, EEG activity and cortical perfusion were significantly reduced in rats receiving LY379268. At 7 days, cortical perfusion was significantly increased in rats treated with LY379268, while EEG activity was depressed as in control rats. While brain edema remained unchanged at 24 h, cortical contusion was significantly decreased by 56% at 7 days after CCI. Cortical glutamate, glucose, and lactate were not influenced. Significant reductions in EEG activity and contusion volume by LY379268 do not appear mediated by attenuated excitotoxicity and energetic impairment. Overall, an additional decrease in cortical perfusion seems to interfere with the anti-edematous potential of LY379268 during the early period following CCI, while an increase in perfusion in LY379268-treated rats at 7 days might contribute to tissue protection.

Working length of locking plates determines interfragmentary movement in distal femur fractures under physiological loading

BACKGROUND This study aimed to investigate the influence of the screw location and plate working length of a locking plate construct at the distal femur on interfragmentary movement under physiological loading. METHODS To quantitatively analyse the influence of plate working length on interfragmentary movements in a locking plate construct bridging a distal femur fracture, a finite element model based on CT (computed tomography) data was physiologically loaded and fracture gap conditions were calculated. Four working lengths with eight screw variations each were systemically analysed. FINDINGS Interfragmentary movements for axial (12-19%, p<0.001) and shear movements (-7.4-545%, p<0.001) at all tested nodes increased significantly with longer plate working length, whereas screw variations within the groups revealed no significant influence. The working length (defined by screw location) dominates the biomechanical fracture gap conditions. INTERPRETATION The current finite element analysis demonstrates that plate working length significantly influences interfragmentary movements, thereby affecting the biomechanical consequences of fracture healing.

Visualization of rat pial microcirculation using the novel orthogonal polarized spectral (OPS) imaging after brain injury

Recently, the novel optical system, orthogonal polarized spectral (OPS) imaging was developed to visualize microcirculation. Investigation of changes in microcirculation is essential for physiological, pathophysiological, and pharmacological studies. In the present study applicability of OPS imaging was assessed to study pial microcirculation in normal and traumatized rat brain. High quality images of rat pial microcirculation in normal and traumatized rats were generated with the OPS imaging, allowing to easily differentiate arterioles and venules with the dura remaining intact. In non-traumatized rats, mean vessel diameter of arterioles and venules of five different cortical regions was 19.1+/-2.7 and 22.2+/-1.4 microm, respectively. In the early phase following focal cortical contusion vessel diameter was significantly decreased in arterioles by 28% while diameter in venules was significantly increased by 27%. For technical reasons velocity in arterioles was not measurable. In venules, mean flow velocity of 0.68+/-0.08 mm/s was significantly decreased by 50% at 30 min after trauma. OPS imaging is an easy to use optical system allowing to generate high quality images and to reliably investigate pial microcirculation without having to remove the dura. This technique opens the possibility to perform longitudinal studies investigating changes in pial microcirculation.