Marek Joukal

Activation of Astrocytes and Microglial Cells and CCL2/CCR2 Upregulation in the Dorsolateral and Ventrolateral Nuclei of Periaqueductal Gray and Rostral Ventromedial Medulla Following Different Types of Sciatic Nerve Injury

Peripheral nerve injuries (PNIs) may result in cellular and molecular changes in supraspinal structures possibly involved in neuropathic pain (NPP) maintenance. Activated glial cells in specific supraspinal subregions may affect the facilitatory role of descending pathways. Sterile chronic compression injury (sCCI) and complete sciatic nerve transection (CSNT) in rats were used as NPP models to study the activation of glial cells in the subregions of periaqueductal gray (PAG) and rostral ventromedial medulla (RVM). Molecular markers for activated astrocytes (glial fibrillary acidic protein, GFAP) and microglial cells (OX42) were assessed by quantitative immunohistochemistry and western blotting. The cellular distribution of CCL2/CCR2 was monitored using immunofluorescence. sCCI induced both mechanical and thermal hypersensitivity from day 1 up to 3 weeks post-injury. Unilateral sCCI or CSNT for 3 weeks induced significant activation of astrocytes bilaterally in both dorsolateral (dlPAG) and ventrolateral PAG (vlPAG) compared to naïve or sham-operated rats. More extensive astrocyte activation by CSNT compared to sCCI was induced bilaterally in dlPAG and ipsilaterally in vlPAG. Significantly more extensive activation of astrocytes was also found in RVM after CSNT than sCCI. The CD11b immunopositive region, indicating activated microglial cells, was remarkably larger in dlPAG and vlPAG of both sides from sCCI- and CSNT-operated rats compared to naïve or sham-operated controls. No significant differences in microglial activation were detected in dlPAG or vlPAG after CSNT compared to sCCI. Both nerve injury models induced no significant differences in microglial activation in the RVM. Neurons and activated GFAP+ astrocytes displayed CCL2-immunoreaction, while activated OX42+ microglial cells were CCR2-immunopositive in both PAG and RVM after sCCI and CSNT. Overall, while CSNT induced robust astrogliosis in both PAG and RVM, microglial cell activation was similar in the supraspinal structures in both injury nerve models. Activated astrocytes in PAG and RVM may sustain facilitation of the descending system maintaining NPP, while microglial activation may be associated with a reaction to long-lasting peripheral injury. Microglial activation via CCR2 may be due to neuronal and astrocytal release of CCL2 in PAG and RVM following injury.

Direct communication of the spinal subarachnoid space with the rat dorsal root ganglia.

The anatomical position of the subarachnoid space (SAS) in relation to dorsal root ganglia (DRG) and penetration of tracer from the SAS into DRG were investigated. We used intrathecal injection of methylene blue to visualize the anatomical position of the SAS in relation to DRG and immunostaining of dipeptidyl peptidase IV (DPP-IV) for detecting arachnoid limiting the SAS. Intrathecal administration of fluorescent-conjugated dextran (fluoro-emerald; FE) was used to demonstrate direct communication between the SAS and DRG. Intrathecal injection of methylene blue and DPP-IV immunostaining revealed that SAS delimited by the arachnoid was extended up to the capsule of DRG in a fold-like recess that may reach approximately half of the DRG length. The arachnoid was found in direct contact to the neuronal body-rich area in the angle between dorsal root and DRG as well as between spinal nerve roots at DRG. Particles of FE were found in the cells of DRG capsule, satellite glial cells, interstitial space, as well as in small and medium-sized neurons after intrathecal injection. Penetration of FE from the SAS into the DRG induced an immune reaction expressed by colocalization of FE and immunofluorescence indicating antigen-presenting cells (MHC-II+), activated (ED1+) and resident (ED2+) macrophages, and activation of satellite glial cells (GFAP+). Penetration of lumbar-injected FE into the cervical DRG was greater than that into the lumbar DRG after intrathecal injection of FE into the cisterna magna. Our results demonstrate direct communication between DRG and cerebrospinal fluid in the SAS that can create another pathway for possible propagation of inflammatory and signaling molecules from DRG primary affected by peripheral nerve injury into DRG of remote spinal segments.

A facial reconstruction and identification technique for seriously devastating head wounds

Many authors have focused on facial identification techniques, and facial reconstructions for cases when skulls have been found are especially well known. However, a standardized facial identification technique for an unknown body with seriously devastating head injuries has not yet been developed. A reconstruction and identification technique was used in 7 cases of accidents involving trains striking pedestrians. This identification technique is based on the removal of skull bone fragments, subsequent fixation of soft tissue onto a universal commercial polystyrene head model, precise suture of dermatomuscular flaps, and definitive adjustment using cosmetic treatments. After reconstruction, identifying marks such as scars, eyebrows, facial lines, facial hair and partly hairstyle become evident. It is then possible to present a modified picture of the reconstructed face to relatives. After comparing the results with photos of the person before death, this technique has proven to be very useful for identifying unknown bodies when other identification techniques are not available. This technique is useful for its being rather quick and especially for its results.

Expression of Regeneration-Associated Proteins in Primary Sensory Neurons and Regenerating Axons After Nerve Injury-An Overview: PROTEINS ASSOCIATED WITH NEURON REGENERATION

Peripheral nerve injury results in profound alterations of the affected neurons resulting from the interplay between intrinsic and extrinsic molecular events. Restarting the neuronal regenerative program is an important prerequisite for functional recovery of the injured peripheral nerve. The primary sensory neurons with their cell bodies in the dorsal root ganglia provide a useful in vivo and in vitro model for studying the mechanisms that regulate intrinsic neuronal regeneration capacity following axotomy. These studies frequently need to indicate the regenerative status of the corresponding neurons. We summarize the critical issues regarding immunohistochemical detection of several regeneration‐associated proteins as markers for the initiation of the regeneration program in rat primary sensory neurons and indicators of axon regeneration in the peripheral nerves. This overview also includes our own results of GAP43 and SCG10 expression in different DRG neurons following double immunostaining with molecular markers of neuronal subpopulations (NF200, CGRP, and IB4) as well as transcription factors (ATF3 and activated STAT3) following unilateral sciatic nerve injury. Anat Rec, 301:1618–1627, 2018.

Fluid content in the pleural cavity protects internal structures against heat

AbstractPurposeIn this paper we present a case report of two people who were found dead in a holiday cottage after a fire. The cause of death in these cases was clarified by the presence of blood in the left pleural cavity in both bodies. MethodsAn inspection of the bodies at the scene of the fire and subsequent examination by a medical examiner suggested the deaths were accidental.ResultsAn autopsy showed that the bodies were those of a man and a woman with severe carbonization of the skin, soft tissues, brain, and some organs of the thoracic and abdominal cavities. There were also isolated stab injuries to the chest, as well as injuries to skeletal structures and intrathoracic organs. The accumulated blood in the pleural cavity had protected the internal organs against thermal destruction and preserved the traumatic findings.ConclusionTo the best of our knowledge this is the first description of the protective effect of hemothorax against thermal damage of the intrathoracic organs.

Fatal Ingestion of Chlumsky Disinfectant Solution

A 32‐year‐old pregnant woman in the 25th week of pregnancy underwent oral glucose tolerance screening at the diabetologists. Later that day, she was found dead in her apartment possibly poisoned with Chlumsky disinfectant solution (solutio phenoli camphorata). An autopsy revealed chemical burns in the digestive system. The lungs and the brain showed signs of severe edema. The blood of the woman and fetus was analyzed using gas chromatography with mass spectrometry and revealed phenol, its metabolites (phenyl glucuronide and phenyl sulfate) and camphor. No ethanol was found in the blood samples. Both phenol and camphor are contained in Chlumsky disinfectant solution, which is used for disinfecting surgical equipment in healthcare facilities. Further investigation revealed that the deceased woman had been accidentally administered a disinfectant instead of a glucose solution by the nurse, which resulted in acute intoxication followed by the death of the pregnant woman and the fetus.

Anatomy of the Human Visual Pathway

Vision is the primary sense in humans. There are approximately one million axons in the optic nerve, constituting almost 40% of the total number of axons in all cranial nerves. The primary sensors for sight are the 130 million rods and seven million cones found in the retina. With the release of glutamate, they transform electromagnetic waves of light with a wavelength between 400 and 700 nm to graded changes of the membrane potential. The signal from photoreceptors continues to the bipolar cells and then to the retinal ganglion cells. Their axons pass through the optic nerve, the optic chiasm, form the optic tract, and reach the lateral geniculate body of the thalamus. The axons coming from the nasal hemiretina are crossed in the optic chiasm, while axons from the temporal hemiretina stay uncrossed. Neurons of the lateral geniculate body send their axons to the optic radiation and terminate in the primary visual cortex – the striate area in the ipsilateral occipital lobe where the first analysis of visual information is performed. Further processing takes place in extrastriate visual areas in the occipital, parietal, and temporal lobes. The visual pathway shows a precise retinotopical organization at all levels that gives the anatomical background for symptoms when some part of optic pathway is damaged.

Biventricular noncompaction cardiomyopathy with malignant arrhythmia as a cause of sudden death

Noncompaction cardiomyopathy (NC) is a rare myocardial disease that belongs to the non-classified congenital cardiomyopathies [1]. NC presentation varies, from being isolated to the left and/or the right ventricle and could be associated with congenital heart defects [2]. The clinical manifestation and morphology of the myocardium is highly variable which can lead to uncertain and even mistaken diagnoses.

Local chemical sympathectomy of rat bone marrow and its effect on marrow cell composition

Existing experimental studies of the effect of sympathetic nerve fibers on bone marrow cells are based on the systemic administration of neurotoxic 6-hydroxydopamine. The method of global chemical sympathectomy has some serious disadvantages and could lead to questionable results. We describe a new method of local chemical sympathectomy of rat femoral bone marrow using guanethidine (Ismelin) delivery using an osmotic mini pump. Local guanethidine treatment for 14days led to complete elimination of sympathetic fibers in femoral bone marrow in contrast to bone marrow of contralateral or naïve femurs. Ablation of sympathetic fibers was associated with a loss of rat endothelial cell marker (RECA) indicating immunophenotype changes in blood vessel endothelial cells, but no significant effect of guanethidine was found on the survival of endothelial cells and mesenchymal stem cells in vitro. Moreover, local guanethidine treatment also elicited a significant reduction of Nestin+/SDF1+ mesenchymal stem cells and c-Kit+/CD90+ hematopoietic stem cells in femoral bone marrow. Tissue-specific chemical sympathectomy of rat bone marrow by guanethidine overcomes some of the drawbacks of systemic administration of neurotoxic compounds like 6-hydroxydopamine and delivers unequivocal evidence on the effects of sympathetic innervation on the cell content of bone marrow.

Cellular reactions of the choroid plexus induced by peripheral nerve injury

The choroid plexus (CP) of brain ventricles forms the blood-cerebrospinal fluid (blood-CSF) barrier that is involved in many diseases affecting the central nervous system (CNS). We used ED1 and ED2 immunostaining to investigate epiplexus cell changes in rat CP after chronic constriction injury (CCI). In contrast to naïve CP, the CP of sham-operated rats showed an increase in the number of ED1+ cells of a similar magnitude during all periods of survival up to 3 weeks, while the number of ED2+ increased only at 3 days from operation. In comparison to naïve and sham-operated animals, the number of ED1+ and ED2+ cells in the epiplexus position increased with the duration of nerve compression. We detected no or negligible cell proliferation in the CP after sham- or CCI-operation. This suggests that increased number of ED1+ and ED2+ cells in the epiplexus position of the CP is derived from peripheral monocytes passing through altered blood-CSF barrier. The changes in epiplexus cells indicate that the CP reacts to tissue injury after the surgical approach itself and that the response to peripheral nerve lesion is greater. This suggests a role for an altered blood-CSF barrier allowing for propagation of signal molecules from damaged tissue and nerve to the CNS.