Cryogenic Injury Research Articles

Utility of Intraoperative Neuromonitoring to Protect against Adjacent Nerve Injury in Musculoskeletal and Lymph Nodal Cryoablation

PurposeTo demonstrate the utility of intraoperative neuromonitoring (IONM) as an effective method of passive thermoprotection against cryogenic injury to neural structures during musculoskeletal and lymph node cryoablation. Material and MethodsTwenty-nine patients (16 men; mean age among men, 68.6 years [range, 45–90 years]; mean age among women, 62.6 years [range, 28–88 years]) underwent 33 cryoablations of musculoskeletal and lymph node lesions. Transcranial electrical motor-evoked potentials (MEPs) and somatosensory-evoked potentials (SSEPs) of target nerves were recorded throughout the ablations. Significant change was defined as waveform amplitude reduction greater than 30% (MEP) and 50% (SSEP). The primary outcomes of this study were immediate postprocedural neurologic deficits and frequency of significant MEP and SSEP amplitude reductions. ResultsSignificant amplitude reductions were detected in 54.5% (18/33) of MEP tracings and 0% (0/33) of SSEP tracings. Following each occurrence of significant amplitude reductions, freeze cycles were promptly terminated. Intraprocedurally, 13 patients had full recovery of amplitudes to baseline, 11 of whom had additional freeze cycles completed. In 5 of 33 (15.2%) cryoablations, there were immediate postprocedural neurologic deficits (moderate adverse events). Unrecovered MEPs conferred a relative risk for neurologic sequela of 23.2 (95% CI, 3.22–167.21; P < .001) versus those with recovered MEPs. All 5 patients had complete neurologic recovery by 12 months. ConclusionsIONM (with MEP but not SSEP) is a reliable and safe method of passive thermoprotection of neurologic structures during cryoablation. It provides early detection of changes in nerve conduction, which when addressed quickly, may result in complete restoration of MEP signals within the procedure and minimize risk of cryogenic neural injury.

Potentiation of Cryogenic Injury of Biological Tissues (Experimental Study)

Potentiation of Cryogenic Injury of Biological Tissues (Experimental Study)

Can delayed grafting of frozen teeth achieve periodontal ligament healing?

Autologous teeth transplanted technology is putting healthy teeth into empty alveolar socket and waiting to be fixed by newly formed alveolar bone and periodontal membrane, to perform chewing function. Vigorous donor teeth are usually impacted teeth or orthodontic excess teeth that are often extracted and discarded during adolescence. However, they should have more potential function that repairing dentition defects due to caries, apical periodontitis, cysts, and other causes after being an adult. Therefore, once the preserved technical is skilled, utilization of dental tissues extracted early in life would create new possibilities. The problem is whether cryopreservation reduces tissue regeneration. Scholars who emphasize periodontal ligament (PDL) healing firmly believe it can avoid pathological reaction and ensure the success of transplantation. Nowadays, the ideal success rate can reach 80.0 % – 91.1%, but the classic transplantation procedure is time-dependent, which requires the donor tooth to be extracted and transplanted immediately. Freezing is a recognized effective way to make physiological and biochemical activities quiescent, and to maintain the potential of cells to relive after rewarming in the same medium. During cells cryopreservation, the challenge is in the inappropriate freezing and thawing condition, intracellular freezing would break cells’ physical structure, spoil normal PDL and increase resorption of root. So even though delay in freezing autotransplantation would greatly expand procedure indications, it has not yet become a mainstream treatment. Slow freezing and vitrification technology are main cryopreservation ways certified effective that essentially prevent ice crystals from damaging cells. To some extent, none of them is perfect considering cell cytotoxicity from cryoprotectants or controlling of freezing rate. Now, with the modified freezing method, we firstly put forward Antifreeze proteins (AFPs) to tooth cryopreservation, and assume that low concentration AFPs combined with vitrification may yield better results in PDL healing of delayed autotransplanted tooth. Based on analyzing the freezing mechanism, cell studies, animal experiments and individual case reports, we propose a procedural freezing method and summarized how to reduce cryogenic injury of cryopreserved tooth for potential use in tooth transplantation. However, more studies and data are needed about masticatory function and long-term prognosis observation.

Habilitation Improves Mouse Gait Development Following Neonatal Brain Injury.

Neonatal brain injury during gait development disrupts neural circuits and causes permanent gait dysfunction. Rehabilitation as an intervention to improve impaired gait function has been used in adults as a treatment for stroke and spinal cord injury. However, although neonates have greater neuroplasticity and regenerative capacity than adults, normal gait development and the effects of habilitation on gait function following neonatal brain injury are largely unknown. In this study, we generated cryogenic injury in mice at postnatal day 2 and subsequently performed habilitative training to promote autonomous limb movement for 4 weeks. We also quantitatively analyzed the gait acquisition process in developing mice using the Catwalk XT system. Using quantitative gait analyses, we showed that during normal gait development in mice, stance phase function matures later than swing phase function. We also demonstrated that habilitation in which active limb movements were enhanced by suspending mice with a rubber band with no floor grounding promotes motor learning, including gait function, in mice with impaired acquisition of gait function resulting from neonatal brain injury. Our findings provide a basis for research on gait development in mice and suggest new habilitation strategies for patients with impaired gait development caused by perinatal brain diseases such as hypoxic-ischemic encephalopathy and periventricular leukomalacia.

Neuro-regenerative imidazole-functionalized GelMA hydrogel loaded with hAMSC and SDF-1α promote stem cell differentiation and repair focal brain injury

Brain tissues that are severely damaged by traumatic brain injury (TBI) is hardly regenerated, which leads to a cavity or a repair with glial scarring. Stem-cell therapy is one viable option to treat TBI-caused brain tissue damage, whose use is, whereas, limited by the low survival rate and differentiation efficiency of stem cells. To approach this problem, we developed an injectable hydrogel using imidazole groups-modified gelatin methacrylate (GelMA-imid). In addition, polydopamine (PDA) nanoparticles were used as carrier for stromal-cell derived factor-1 (SDF-1α). GelMA-imid hydrogel loaded with PDA@SDF-1α nanoparticles and human amniotic mesenchymal stromal cells (hAMSCs) were injected into the damaged area in an in-vivo cryogenic injury model in rats. The hydrogel had low module and its average pore size was 204.61 ± 41.41 nm, which were suitable for the migration, proliferation and differentiation of stem cells. In-vitro cell scratch and differentiation assays showed that the imidazole groups and SDF-1α could promote the migration of hAMSCs to injury site and their differentiation into nerve cells. The highest amount of nissl body was detected in the group of GelMA-imid/SDF-1α/hAMSCs hydrogel in the in-vivo model. Additionally, histological analysis showed that GelMA-imid/SDF-1α/hAMSCs hydrogel could facilitate the regeneration of regenerate endogenous nerve cells. In summary, the GelMA-imid/SDF-1α/hAMSCs hydrogel promoted homing and differentiation of hAMSCs into nerve cells, and showed great application potential for the physiological recovery of TBI.

Subglottic Cryotherapy: A Pilot Study in New Zealand White Rabbits

To describe proof of concept and pilot data for a cryotherapy application in the subglottis in a rabbit airway model. Four New Zealand white rabbits (3 experimental, 1 control) underwent general anesthesia and laryngoscopy and bronchoscopy. Experimental animals had cryotherapy applied with a direct contact 1.9-mm cryoprobe. Animals were euthanized at days 0, 2, and 6 posttreatment. Histologic changes were assessed in the treated subglottic tissues. This preliminary work has demonstrated that, with early cryogenic injury in the subglottis, there is mild mucosal epithelial injury associated with submucosal edema, acute inflammatory infiltrate, and degeneration of venule endothelial cells. Mucosal epithelial repair and resolution of the inflammatory response appear to be relatively rapid. We hope that this may provide a foundation to further explore cryotherapy as a primary or adjuvant treatment option for pediatric subglottis stenosis.

Roles of Reelin/Disabled1 pathway on functional recovery of hemiplegic mice after neural cell transplantation; Reelin promotes migration toward motor cortex and maturation to motoneurons of neural grafts

Reelin is a large glycoprotein which regulates central nervous system (CNS) development. Dysfunctions of Reelin were reported on certain neuropsychiatric diseases. We examined involvement of Reelin pathway in functional recovery of hemiplegic mice after neural transplantation.Reelin was expressed 1 day after cryogenic injury of right motor cortex. We transplanted neural stem/progenitor cells (NSPCs) from wild-type mice into ipsilateral striatum of hemiplegic mice. The grafts migrated from the striatum and reached the injured cortex 14 days after transplantation. The transplantation significantly improved their motor functions (P < .05).The NSPCs migrating toward the cortex expressed Reelin receptors, Apoer and Vldlr, and phosphorylated Disabled1 (Dab1), a downstream signaling molecule of Reelin. The grafts expressed Ncadherin and active form of Integrin β1, both of which were known to become active with Reelin stimulation. At day 28, the grafts expressed Ctip2, Crim1, Foxp2, and Fezf2, all of which were forebrain motoneuron associated markers, and Nfm and Synapsin1 on the damaged cortex.We then transplanted NSPCs of yotari mice (yot/yot genotype) having nonfunctional Dab1 by a mutation of its gene. Majority of the grafts from yotari mice (>80%) did not migrate and thus remained at the striatum. The grafts did not express the forebrain motoneuron associated markers nor the cell adhesion molecules including Ncadherin and active Integrin β1.Reelin pathway was involved in graft migration by regulating certain adhesion molecules and in their differentiation to functional motoneurons accompanying synapse formation. We suggested involvement of Reelin pathway for neural regeneration and functional recovery of hemiplegic mice in adulthood after neural transplantation.

Allyl isothiocyanate attenuates oxidative stress and inflammation by modulating Nrf2/HO-1 and NF-κB pathways in traumatic brain injury in mice.

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in young adults and children in the industrialized countries; however, there are presently no FDA approved therapies. TBI results in oxidative stress due to the overproduction of reactive oxygen species and overwhelming of the endogenous antioxidant mechanisms. Recently, it has been reported that antioxidants including phytochemicals have a protective role against oxidative damage and inflammation after TBI. To analyze the effects of a naturally occurring antioxidant molecule, allyl isothiocyanate (AITC), on the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor kappa B (NF-κB) signaling pathways in TBI, a cryogenic injury model was induced in mice. Here, we showed that AITC administered immediately after the injury significantly decreased infarct volume and blood-brain barrier (BBB) permeability. Protein levels of proinflammatory cytokines interleukin-1β (IL1β) and interleukin-6 (IL6), glial fibrillary acidic protein (GFAP) and NF-κB were decreased, while Nrf2, growth-associated protein 43 (GAP43) and neural cell adhesion molecule levels were increased with AITC when compared with vehicle control. Our results demonstrated that the antioxidant molecule AITC, when applied immediately after TBI, provided beneficial effects on inflammatory processes while improving infarct volume and BBB permeability. Increased levels of plasticity markers, as well as an antioxidant gene regulator, Nrf2, by AITC, suggest that future studies are warranted to assess the protective activities of dietary or medicinal AITC in clinical studies.

Analysis of the protective effects of a neuronal Cav2.1 calcium channel in brain injury

We previously reported that rolling Nagoya mice carrying a mutation in the α1 subunit of the Cav2.1 channel protective from ischemia- and kainate-induced neuronal damage. However, the protective effect of this mutation and its relationship to brain injury recovery have not been examined. To examine the relationship between Cav2.1 channel function and brain injury, we induced cryogenic brain damage in homozygous rolling Nagoya (rol/rol), control wild-type (+/+), ω-agatoxin IVA-pretreated +/+ (ω-aga +/+), and ω-agatoxin IVA-post-treated +/+ (ω-aga-post-treated +/+) mice. We measured the lesion area, blood brain–barrier permeability and performed immunohistochemistry and western blot analysis. The lesions of rol/rol and ω-aga +/+ mice were significantly smaller than those observed in +/+ mice at both day 1 and day 7 after injury. Similar results were shown in blood–brain barrier permeability. We observed more reactive astrogliosis in +/+ mice than in rol/rol or ω-aga +/+ mice. rol/rol and ω-aga +/+ mice had fewer degenerating cells due to cryogenic injury than did +/+ mice at both day 1 and day 7. ω-Aga-post-treated +/+ mice 24h after injury were sacrificed on day 7. The lesions were smaller in ω-aga-post-treated +/+ mice than those in vehicle-treated +/+ mice. We also examined phosphorylated p38 (pp38) at the injured site. ω-Aga-post-treated +/+ mouse brain slices showed weak pp38 signal; vehicle-treated +/+ mouse brain slices were pp38-positive. These findings demonstrate that the mutant Cav2.1 channel exerts a protective effect against cryogenic brain injury in rolling Nagoya mice. Our results indicate that inhibitors of the Cav2.1-dependent p38 signaling cascade would be useful as therapeutic agents in the treatment of brain injury.

Next generation design, development, and evaluation of cryoprobes for minimally invasive surgery and solid cancer therapeutics: in silico and computational studies.

Cryosurgery is a widely regarded minimally invasive surgery for treatment of various types of cancers. It involves destruction of cancer cells within a limited spatial domain by exposing them to very low temperatures while minimizing injury to surrounding peripheral healthy tissues. Surprisingly, despite increasing demands for cryosurgery, there has been limited innovation in the design of cryoprobes, particularly in solid tumors (e.g., breast, prostate, and lung cancers). For advances in cancer therapeutics, integrative biology research can illuminate the mechanistic interface between a surgical cryoprobe and its tissue site of action. Here, we describe the design and development of three novel low pressure liquid nitrogen (LN2) cryoprobes with different physical dimensions and the parameters that determine their effectiveness experimentally, using water and bio-gel as the phase changing mediums. Smaller diameter low pressure probes produced lesser cryogenic injury. Vapor Separator is found to be an effective means (particularly for smaller diameter probes) to remove the vapor lock in the LN2 low pressure cryoprobes and also to reduce the precooling time. The low pressure LN2 cryoprobes produced lower probe temperatures and consequently larger and faster iceball growth for low cooling loads. Additionally, a numerical code was written in MATLAB based on the Enthalpy method to simulate the bio-heat transfer in a cryosurgical process. The numerical code is validated by analytical solution, laboratory experiments, and data from an in vivo cryosurgery. The developed numerical code is presented herein to illustrate that LN2 cryoprobes capable of producing lower probe temperatures produce more efficient cryosurgical operation by reducing the buffer zone and duration of surgery.This is the first report, to the best of our knowledge, on design of the next generation of LN2 surgical cryoprobes. These new surgical cryoprobes offer potentials for future preclinical and clinical testing in solid cancers.

Regional difference of reactive astrogliosis following traumatic brain injury revealed by hGFAP-GFP transgenic mice

Reactive astrocytes greatly influence the wound healing and neuronal regeneration processes following brain injury. However, the origin and fate of reactive astrocytes appear to be different depending on the type, severity and duration of brain injury. Using the cryogenic traumatic brain injury model, here we comprehensively addressed the regional differences of reactive astrocytes in the injured cortex. In the proximal region of injury site, NG2-expressing and cytoplasmic Olig2-labeled cells were densely localized 3 days after the injury. Next to this proximal layer, most of reactive astrocytes did not express NG2 but exhibited radial glia-like shape with elongated processes. Accordingly, they expressed the progenitor or radial glial markers, such as vimentin, brain lipid binding protein (BLBP) and the green fluorescent protein (GFP) under the control of the human GFAP (hGFAP) promoter. However, only few glial fibrillary acidic protein (GFAP) expressing astrocytes were found in this layer. Distal to the injury site, most of astrocytes strongly expressed GFAP with hypertonic morphology. At day 15 after injury, all layers expressing GFAP and other marker expressions disappeared, indicating the termination of reactive astrogliosis. Taken together, our data suggest that reactive astrogliosis occurs in a regionally segregated manner in the early phase of brain injury.

Effect of laser phototherapy on wound healing following cerebral ischemia by cryogenic injury

Laser phototherapy emerges as an alternative or auxiliary therapy for acute ischemic stroke, traumatic brain injury, degenerative brain disease, spinal cord injury, and peripheral nerve regeneration, but its effects are still controversial. We have previously found that laser phototherapy immunomodulates the response to focal brain damage. Following direct cortical cryogenic injury the effects of laser phototherapy on inflammation and repair was assessed after cryogenic injury (CI) to the central nervous system (CNS) of rats. The laser phototherapy was carried out with a 780 nm AlGaAs diode laser. The irradiation parameters were: power of 40 mW, beam area of 0.04 cm 2, energy density of 3 J/cm 2 (3 s) in two points (0.12 J per point). Two irradiations were performed at 3 h-intervals, in contact mode. Rats (20 non-irradiated – controls and 20 irradiated) were used. The wound healing in the CNS was followed in 6 h, 1, 7 and 14 days after the last irradiation. The size of the lesions, the neuron cell viability percentages and the amount of positive GFAP labeling were statistically compared by ANOVA complemented by Tukey’s test ( p < 0.05). The distribution of lymphocytes, leukocytes and macrophages were also analyzed. CI created focal lesions in the cortex represented by necrosis, edema, hemorrhage and inflammatory infiltrate. The most striking findings were: lased lesions showed smaller tissue loss than control lesions in 6 h. During the first 24 h the amount of viable neurons was significantly higher in the lased group. There was a remarkable increase in the amount of GFAP in the control group by 14 days. Moreover, the lesions of irradiated animals had fewer leukocytes and lymphocytes in the first 24 h than controls. Considering the experimental conditions of this study it was concluded that laser phototherapy exerts its effect in wound healing following CI by controlling the brain damage, preventing neuron death and severe astrogliosis that could indicate the possibility of a better clinical outcome.

Low Hematocrit Levels Increase Intracranial Pressure in an Animal Model of Cryogenic Brain Injury: Erratum

Low Hematocrit Levels Increase Intracranial Pressure in an Animal Model of Cryogenic Brain Injury: Erratum

Upregulation of the P2X7 receptor after cryogenic injury to rat brain

The P2X7 receptor is a member of the P2X family of adenosine triphosphate-gated ion channels. It is expressed in the central nervous system and is associated with several pathological conditions. Here, we examined the expression of P2X7 with immunohistochemistry after cryogenic injury to the rat cortex. One day after cortical cryogenic injury, P2X7 immunoreactive cells were increased in regions surrounding the cold-injured site. These P2X7 positive cells were also immunoreactive for OX42, a microglial marker, and possessed the short, thick processes that are characteristic of activated microglia. These results suggest that the increased local expression of P2X7 may be associated with microglial activation, contributing to detrimental and/or protective functions around the cold-injured site.

Surface dialysis after experimental brain injury: modification of edema fluid flow in the rat model

This study was undertaken to determine if dialysis of damaged brain surface can reduce cerebrospinal fluid (CSF) pressure and progressive brain edema. The authors secondarily determined if local brain cooling was simultaneously possible. Telemetric pressure transmitters were implanted into the lumbar subarachnoid space of 58 young adult male rats. Cryogenic brain injury was created and 2 hours later decompressive craniectomy was performed. An osmotic cell with a semipermeable dialysis membrane placed on the damaged brain surface was perfused with dextran 15% solution for 2 or 4 hours. Water content was determined in the cerebral hemispheres using the wet-dry weight method. Evans blue-albumin spread was measured morphometrically. Brain temperature was measured bilaterally. The CSF pressure increased after cryogenic injury and decreased after craniotomy. Two hours of brain dialysis significantly reduced CSF pressure in comparison with craniotomy alone and sham dialysis. Injured brain had higher water content, but this was not affected by dialysis. Spread of Evans blue-albumin, however, was significantly reduced by the treatment. Cooling of the dialysis solution caused significant local brain cooling. Surface dialysis of cryogenically injured rat brain controls CSF pressure and reduces intraparenchymal spread of edema fluid in the acute period after injury. The authors postulate that edema fluid moves into the osmotic cell rather than spreading through the uninjured brain. Long-term experiments will be needed to prove that this combination therapy is effective.

Salvage cryotherapy for recurrent prostate cancer after radiation failure: current status and future perspectives

In the last decade, cryotherapy has emerged as a minimally invasive alternative for the treatment of localized or locally advanced prostate cancer. With better understanding of the pathophysiology of cryogenic injury and the introduction of the gas-driven systems, the procedure has been optimized to improve patient survival and reduce complication. The results of salvage cryotherapy are becoming more promising. Modifying standard cryotherapy technique by the use of a urethral warming catheter and thermocouples has resulted in the reduction in serious complications and improved quality of life following the procedure. Focal nerve-sparing cryotherapy represents a novel approach to treat prostate cancer and preserve potency. Cryoimmunotherapy and cryochemotherapy are new concepts in adjuvant therapy. Early in vitro studies revealed the feasibility of such an approach. Further clinical trials are essential before using these models as standard treatment.

Neuroprotection by erythropoietin administration after experimental traumatic brain injury

A large body of evidence indicates that the hormone erythropoietin (EPO) exerts beneficial effects in the central nervous system (CNS). To date, EPO's effect has been assessed in several experimental models of brain and spinal cord injury. This study was conducted to validate whether treatment with recombinant human EPO (rHuEPO) would limit the extent of injury following experimental TBI. Experimental TBI was induced in rats by a cryogenic injury model. rHuEPO or placebo was injected intraperitoneally immediately after the injury and then every 8 h until 2 or 14 days. Forty-eight hours after injury brain water content, an indicator of brain edema, was measured with the wet-dry method and blood-brain barrier (BBB) breakdown was evaluated by assay of Evans blue extravasation. Furthermore, extent of cerebral damage was assessed. Administration of rHuEPO markedly improved recovery from motor dysfunction compared with placebo group (P<0.05). Brain edema was significantly reduced in the cortex of the EPO-treated group relative to that in the placebo-treated group (80.6+/-0.3% versus 91.8%+/-0.8% respectively, P<0.05). BBB breakdown was significantly lower in EPO-treated group than in the placebo-treated group (66.2+/-18.7 mug/g versus 181.3+/-21 mug/g, respectively, P<0.05). EPO treatment reduced injury volume significantly compared with placebo group (17.4+/-5.4 mm3 versus 37.1+/-5.3 mm3, P<0.05). EPO, administered in its recombinant form, affords significant neuroprotection in experimental TBI model and may hold promise for future clinical applications.

The MRL mouse repairs both cryogenic and ischemic myocardial infarcts with scar

The MRL mouse strain shows extraordinary wound healing capacities. Some years ago, Leferovich et al. (Proc Natl Acad Sci U S A 2001;98:9830-35) have reported the absence of scar formation after cryogenically-induced right ventricular myocardial infarcts in adult MRL mice. An independent group (Oh et al., Cardiovasc Pathol 2004;13:203-6) found that MRL mice repair left ventricular ischemic infarcts after coronary artery ligation with regular scar formation. Given the divergent outcomes in infarct healing in MRL mice reported by those two studies, we have investigated whether MRL mice heal myocardial infarcts without scar both in the cryoinjury and in the coronary ligation model. Four different protocols of cryogenically induced right and left ventricular injury, as well as permanent ligation of the left anterior descending coronary artery, were tested in adult MRL and control C57Bl/6 mice. At 60 days after experimental infarction, MRL mice showed pronounced scarring of the affected right and left ventricular areas, with no significant differences in infarct size and thickness between MRL and C57Bl/6 mice using any of the five experimental protocols. Analysis of cell proliferation by 5-bromo-2'-deoxyuridine (BrdU) incorporation into the DNA did not show any difference between the two strains of mice after infarction. Histological analysis of infarct areas using picrosirius red staining did not show differences in extent of collagen and distribution between the two mouse strains. MRL mice heal myocardial infarcts with scar formation in response to ischemic as well as to cryogenic injuries.

Mouse brains deficient in neuronal PDGF receptor‐β develop normally but are vulnerable to injury

Platelet-derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null-knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the beta subunit of PDGFR (PDGFR-beta) was genetically deleted in CNS neurons to elucidate the role of PDGFR-beta, particularly in the post-natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR-beta detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild-type mice was depleted, but PDGFR-beta detected in blood vessels remained unaffected. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT-mediated dUTP-biotin nick end labeling (TUNEL)-positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR-beta expressed in neurons protects them from cryogenic injury and NMDA-induced excitotoxicity.

Specificity and divergence in the neurobiologic effects of different metallothioneins after brain injury

Brain injury and neuroinflammation are pathophysiologic contributors to acute and chronic neurologic disorders, which are progressive diseases not fully understood. Mammalian metallothioneins I and II (MT-I&II) have significant neuroprotective functions, but the precise mechanisms underlying these effects are still unknown. To gain insight in this regard, we have evaluated whether a distant, most likely single-domain MT (Drosophila MTN) functions similarly to mammalian MT-I&II (recombinant mouse MT-I and human MT-IIa and native rabbit MT-II) after cryogenic injury to the cortex in Mt1&2 KO mice. All the recombinant proteins showed similar neuroprotective properties to native MT-II, significantly reducing brain inflammation (macrophages, T cells, and pro-inflammatory cytokines), oxidative stress, neurodegeneration, and apoptosis. These results in principle do not support specific protein-protein interactions as the mechanism underlying the neuroprotective effects of these proteins because a non-homologous and structurally unrelated MT such as Drosophila MTN functions similarly to mammalian MTs. We have also evaluated for the first time the neurobiologic effects of exogenous MT-III, a major CNS MT isoform. Human rMT-III, in contrast to human nMT-IIa, did not affect inflammation, oxidative stress, and apoptosis, and showed opposite effects on several growth factors, neurotrophins, and markers of synaptic growth and plasticity. Our data thus highlight specific and divergent roles of exogenous MT-III vs. the MT-I&II isoforms that are consistent with those attributed to the endogenous proteins, and confirm the suitability of recombinant synthesis for future therapeutic use that may become relevant to clinical neurology.