Vitrification preserves murine and human donor cells for generation of tissue-engineered intestine

Background

Short bowel syndrome causes significant morbidity and mortality. Tissue-engineered intestine may serve as a viable replacement. Tissue-engineered small intestine (TESI) has previously been generated in the mouse model from donor cells that were harvested and immediately reimplanted; however, this technique may prove impossible in children who are critically ill, hemodynamically unstable, or septic. We hypothesized that organoid units (OU), multicellular clusters containing epithelium and mesenchyme, could be cryopreserved for delayed production of TESI.

Methods

OU were isolated from <3 wk-old mouse or human ileum. OU were then cryopreserved by either standard snap freezing or vitrification. In the snap freezing protocol, OU were suspended in cryoprotectant and transferred directly to −80°C for storage. The vitrification protocol began with a stepwise increase in cryoprotectant concentration followed by liquid supercooling of the OU solution to −13°C and nucleation with a metal rod to induce vitrification. Samples were then cooled to −80°C at a controlled rate of −1°C/min and subsequently plunged into liquid nitrogen for long-term storage. OU from both groups were maintained in cryostorage for at least 72 h and thawed in a 37°C water bath. Cryoprotectant was removed with serial sucrose dilutions and OU were assessed by Trypan blue assay for post-cryopreservation viability. Via techniques previously described by our laboratory, the thawed murine or human OU were either cultured in vitro or implanted on a scaffold into the omentum of a syngeneic or irradiated Nonobese Diabetic/Severe Combined Immunodeficiency, gamma chain deficient adult mouse. The resultant TESI was analyzed by histology and immunofluorescence.

Results

After cryopreservation, the viability of murine OU was significantly higher in the vitrification group (93 ± 2%, mean ± standard error of the mean) compared with standard freezing (56 ± 6%) (P < 0.001, unpaired t-test, n = 25). Human OU demonstrated similar viability after vitrification (89 ± 2%). In vitro culture of thawed OU produced expanding epithelial spheres supported by a layer of mesenchyme. TESI was successfully generated from the preserved OU. Hematoxylin and eosin staining demonstrated a mucosa composed of a simple columnar epithelium whereas immunofluorescence staining confirmed the presence of both progenitor and differentiated epithelial cells. Furthermore, beta-2-microglobulin confirmed that the human TESI epithelium originated from human cells.

Conclusions

We demonstrated improved multicellular viability after vitrification over conventional cryopreservation techniques and the first successful vitrification of murine and human OU with subsequent TESI generation. Clinical application of this method may allow for delayed autologous implantation of TESI for children in extremis.     

Rats recovering from unilateral barrel-cortex ischemia are capable of completing a whisker-dependent task using only their affected whiskers

Rats use their vibrissae for a variety of exploratory tasks including location of objects and discrimination of texture. This study examines recovery in vibrissal function following a unilateral ischemic injury to the somatosensory cortex. Vibrissal function was examined in adult food-restricted rats performing on a two-texture discrimination device. Animals were trained and tested until the criteria of >80% correct choices was demonstrated on three consecutive days. Ischemic rats were constrained to use the affected whiskers by clipping the ipsilateral vibrissae. One group was tested after ischemia, a second group was trained before ischemia and then tested, and a third group was pre-trained and received whisker stimulation and tested post-ischemia. Nai;ve animals recovering from ischemia took longer to reach criteria than intact or unilateral trimmed control animals. Pre-trained animals with compression ischemia receiving whisker stimulation with sucrose water completed the task to criteria in the fewest number of trials. The results indicate that recovery of vibrissal function occurs following a unilateral ischemic injury. Histological analysis in animals without whisker stimulation indicates that the number of normal appearing cortical barrels following ischemia was inversely correlated to the number of trials needed to complete the behavioral task. This suggests that the natural recovery of the ability to discriminate textures is related to the degree of damage to the barrel cortex. The relationship between cortical barrels and behavioral recovery did not hold for the ischemic animals receiving whisker stimulation. This latter group demonstrated recovery despite marked anatomical lesions suggesting that the intervention influenced reorganization.     

Epileptic spasms and partial seizures as a single ictal event

PURPOSE: To investigate the phenomenon of epileptic spasms (ESs) associated with other seizure types in a single ictal event and to study the predictive value of this phenomenon regarding etiology and prognosis. METHODS: We selected retrospectively eight female and five male patients, who had ESs and other seizure types within a single seizure event and for whom a video-EEG recording of the phenomenon was performed in at least one situation. RESULTS: The seizure type associated with ESs was a partial seizure in all patients. We identified three groups with different seizure patterns regarding the temporal association of ES and partial seizures (PSs): (a) PS followed by ES; (b) PS appearing during a cluster of ESs without interrupting the cluster; and (c) complex seizure interaction with a succession of ESs and PSs in a close but variable temporal association. Underlying disorders included cortical dysplasia (three patients), complex cerebral malformations (two patients), and perinatal anoxic-ischemic injuries (two patients); four cases were classified as cryptogenic, and in two children, etiology was unknown, but prenatal origin was suspected. Outcome was poor in nine cases with intractable epilepsy; four cases had a favorable outcome, defined as complete cessation of epileptic seizures. CONCLUSIONS: The phenomenon of associated ESs and PSs as a single ictal event can be related to different etiologies and should not be considered distinctive for cortical malformations or severe brain damage. Different seizure patterns of associated ESs and PSs provide no hint for etiology or prognosis. Outcome is prevalently but not constantly unfavorable in patients with the phenomenon.     

"Beware of coming too close to me"--development-oriented psychotherapy of a dangerous aggressive boy with early complex trauma experiences

Early maltreatment of children can lead to severe disorders in the regulation of behaviour and affect, alterations in awareness and distorted perception. In development-oriented psychotherapy, therapeutic interventions which are directed to regulating processes, decentration or mentalization, desomatization and symbolization of enacted messages play a central role. The results of early traumatization and of therapeutic interventions are portrayed, examining the multi-dimensional diagnosis and therapy of a dangerously aggressive 12-year-old boy as an example. From this, it becomes clear how, through new experiences with regulating others, he gradually surfaces from a world of annihilation and destruction and learns to survive and to live.     

Mechanisms of peripheral microvascular dysfunction in transgenic mice overexpressing the Alzheimer's disease amyloid Abeta protein

Freshly prepared soluble amyloid (Abeta) peptide has been reported to have vascular actions both in vitro and in vivo. This study was designed to examine the in vivo microvascular effects of beta in two skin microvascular model systems that might reflect possible short and long-term vascular effects of this peptide. Short-term vascular effects were examined using freshly prepared soluble Abeta(1-40) peptide superfused over naive rat skin microvasculature for 15 min. Peripheral microvascular functional changes in 9-months-old transgenic (Tg) mice overexpressing soluble beta in the brain, peripheral circulation and other tissues, were also examined. Microvascular responses were monitored using laser Doppler flowmetry from the base of a blister raised on the hind footpad of the animals. Endothelial-dependent and independent vasodilatation responses (VD) were examined using acetylcholine (ACh) and sodium nitroprusside (SNP) respectively. The exposure of na?ve rat skin microvasculature to Abeta(1-40) resulted in an immediate vasoconstriction (VC) that prevented ACh but not SNP from inducing a subsequent VD response. The vascular effects of Abeta(1-40) were reversed by antioxidants (superoxide dismutase and catalase) and an endothelin A (ETA) receptor antagonist (BQ-123). Tg mice overexpressing soluble Abeta and C100 showed significant reductions in both endothelial-dependent and endothelial-independent VD that were also reversed by antioxidants and BQ-123. In conclusion, this study provided evidence to support the notion of peripheral vascular effects of Abeta in vivo and present novel evidence for alterations in endothelial and smooth muscle cell function in peripheral skin microvasculature in Tg mice overexpressing Abeta and C100. We suggest that skin microvasculature is a useful model to examine the mechanisms underlying the vascular actions of the Abeta protein.     

Experiments on effects of an intermittent 16.7-Hz magnetic field on salivary melatonin concentrations, rectal temperature, and heart rate in humans

OBJECTIVES: The present experiments concerned the hypothesis that an intermittent, strong and extremely low frequency magnetic field reduces salivary melatonin levels and delays consecutively the nadirs of rectal temperature and heart rate. METHODS: Twelve healthy young men (18-25 years) participated in three randomly permuted sessions, which were performed as constant routines. The participants kept a strict bed rest over 26 h, air temperature was 20 degrees C, illumination < 30 lx, and sound level < 50 dBA. Salivary melatonin levels were determined hourly, rectal temperature and heart rate were registered continuously throughout. An intermittent magnetic field was administered in one session from 6 p.m. to 2 a.m. at 16.7 Hz, 0.2 mT and alternating on/off-periods of 15 s. This situation was compared with a control session without any additional stress. Another session was performed to determine the participants' ability to respond to a well-known melatonin-suppressing stress, namely bright light (1,500 lx, 10 p.m.-2 a.m.). RESULTS: Bright light inhibited melatonin synthesis in all 12 participants and delayed the nadirs of rectal temperature and heart rate. The only significant alteration that was associated with exposure to the magnetic field was a delay in the heart rate nadir, which was not mediated by an accordingly altered melatonin profile. CONCLUSION: The fact that the circadian rhythm of only the heart rate was altered indicates an internal dissociation which might constitute a health risk in the long run and needs to be investigated more extensively.