Exploration and validation of m7G-related genes as signatures in the immune microenvironment and prognostic indicators in low-grade glioma.

OBJECTIVES: Currently, an increasing number of studies are focusing on the impact of m7G modification in cancer. This study aims to investigate the prognostic value of m7G-related genes in low-grade glioma (LGG). METHODS: LGG samples were obtained from the CGGA database, and normal samples were obtained from GTEx. Differentially expressed m7G-related genes were identified, and genes highly associated with macrophage M2 in LGG patients were identified by immuno-infiltration and WGCNA analysis. The intersection of differentially expressed m7G-related genes and macrophage M2-associated genes yielded candidate genes, and hub genes were identified using 5 algorithms in CytoHubba. Enrichment analysis verified the relevant pathways of hub genes, and their performance in tumor classification was evaluated. RESULTS: A total of 3329 differentially expressed m7G-related genes were identified. 1289 genes were highly associated with macrophage M2 in LGG patients. The intersection of m7G-related genes and results in WGCNA yielded 840 candidate genes, and six hub genes (STXBP1, CPLX1, PAB3A, APBA1, RIMS1, and GRIN2B) were identified. Hub genes were enriched in synaptic transmission-related pathways and showed good performance for tumor classification. There were significant differences in survival levels between clusters. CONCLUSIONS: The identified m7G-related genes may provide new insight into the treatment and prognosis of LGG.

Fibrosis Distinguishes Critical Limb Ischemia Patients from Claudicants in a Transcriptomic and Histologic Analysis.

Most patients with critical limb ischemia (CLI) from peripheral arterial disease (PAD) do not have antecedent intermittent claudication (IC). We hypothesized that transcriptomic analysis would identify CLI-specific pathways, particularly in regards to fibrosis. Derivation cohort data from muscle biopsies in PAD and non-PAD (controls) was obtained from the Gene Expression Omnibus (GSE120642). Transcriptomic analysis indicated CLI patients (N = 16) had a unique gene expression profile, when compared with non-PAD controls (N = 15) and IC (N = 20). Ninety-eight genes differed between controls and IC, 2489 genes differed between CLI and controls, and 2783 genes differed between CLI and IC patients. Pathway enrichment analysis showed that pathways associated with TGFß, collagen deposition, and VEGF signaling were enriched in CLI but not IC. Receiver operating curve (ROC) analysis of nine fibrosis core gene expression revealed the areas under the ROC (AUC) were all >0.75 for CLI. Furthermore, the fibrosis area (AUC = 0.81) and % fibrosis (AUC = 0.87) in validation cohort validated the fibrosis discrimination CLI from IC and control (all n = 12). In conclusion, transcriptomic analysis identified fibrosis pathways, including those involving TGFß, as a novel gene expression feature for CLI but not IC. Fibrosis is an important characteristic of CLI, which we confirmed histologically, and may be a target for novel therapies in PAD.

Caspase1/11 signaling affects muscle regeneration and recovery following ischemia, and can be modulated by chloroquine.

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia.

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3-/-) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1ß (IL-1ß), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3-/- mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.

Chloroquine improves the response to ischemic muscle injury and increases HMGB1 after arterial ligation.

OBJECTIVE: We have previously shown that exogenous administration of the nuclear protein high mobility group box 1 (HMGB1) improves angiogenesis after tissue ischemia. Antagonizing HMGB1 prolongs muscle necrosis and deters regeneration. In this study, we evaluated HMGB1 expression in peripheral arterial disease (PAD) and the mechanisms that promote its release in a murine model of hindlimb ischemia. Specifically, we investigated how chloroquine (CQ), a commonly employed disease-modifying antirheumatic drug, promotes HMGB1 release from muscle. We hypothesized that CQ could increase HMGB1 locally and systemically, allowing it to mediate recovery from ischemic injury. METHODS: Muscle biopsies were performed on patients undergoing lower extremity surgery for non-PAD-related disease as well as for claudication and critical limb ischemia. Clinical symptoms and ankle-brachial indices were recorded for each patient. HMGB1 was detected in muscle sections using immunohistochemical staining. Unilateral femoral artery ligation was performed on both wild-type and inducible HMGB1 knockout mice. Wild-type mice were administered intraperitoneal CQ 2 weeks before and after femoral artery ligation. Laser Doppler perfusion imaging was used to determine perfusion recovery. Serum and tissue levels of HMGB1 were measured at designated time points. In vitro, cultured C2C12 myoblasts were treated with increasing doses of CQ. HMGB1, autophagosome formation, p62/SQSTM1 accumulation, caspase-1 expression and activity, and lactate dehydrogenase levels were measured in supernatants and cell lysates. RESULTS: Nuclear expression of HMGB1 was prominent in patients with claudication and critical limb ischemia (P < .05) compared with controls. CQ-treated mice had elevated serum HMGB1 and diffuse HMGB1 staining in muscle (P < .01). In wild-type mice, CQ treatment resulted in higher laser Doppler perfusion imaging ratios in the ischemic limb at 7 days (P < .03) and less fat replacement after 2 weeks (P < .03). In cultured myoblasts, CQ induced autophagosome accumulation, inhibited p62/SQSTM-1 degradation, and activated caspase-1. CONCLUSIONS: HMGB1 is prominently expressed in PAD muscle but mostly confined to the nucleus. Our in vivo data suggest that HMGB1 mobilization into the sarcoplasm and serum can be increased with CQ, possibly through caspase-1-mediated pathways. Whereas HMGB1 can be released by many cell types, these studies suggest that the muscle may be an important additional source that is relevant in PAD.

The NLRP3 inflammasome and bruton's tyrosine kinase in platelets co-regulate platelet activation, aggregation, and in vitro thrombus formation.

Cleavage of interleukin-1ß (IL-1ß) is a key inflammatory event in immune cells and platelets, which is mediated by nucleotide-binding domain leucine rich repeat containing protein (NLRP3)-dependent activation of caspase-1. In immune cells, NLRP3 and caspase-1 form inflammasome complexes with the adaptor proteins apoptosis-associated speck-like protein containing a CARD (ASC) and bruton's tyrosine kinase (BTK). In platelets, however, the regulatory triggers and the functional effects of the NLRP3 inflammasome are unknown. Here, we show in vitro that the platelet NLRP3 inflammasome contributes to platelet activation, aggregation, and thrombus formation. NLRP3 activity, as monitored by caspase-1 activation and cleavage and secretion of IL-1ß, was upregulated in activated platelets, which was dependent on platelet BTK. Pharmacological inhibition or genetic ablation of BTK in platelets led to decreased platelet activation, aggregation, and in vitro thrombus formation. We identify a functionally relevant link between BTK and NLRP3 in platelets, with potential implications in disease states associated with abnormal coagulation and inflammation.

TLR4 Deters Perfusion Recovery and Upregulates Toll-like Receptor 2 (TLR2) in Ischemic Skeletal Muscle and Endothelial Cells.

Toll-like receptors (TLRs) play an important role in regulating muscle regeneration and angiogenesis in response to ischemia. TLR2 knockout mice exhibit pronounced skeletal muscle necrosis and abnormal vessel architecture after femoral artery ligation, suggesting that TLR2 signaling is protective during ischemia. TLR4, an important receptor in inflammatory signaling, has been shown to regulate TLR2 expression in other systems. We hypothesize that a similar relationship between TLR4 and TLR2 may exist in hindlimb ischemia in which TLR4 upregulates TLR2, a mediator of angiogenesis and perfusion recovery. We examined the expression of TLR2 in unstimulated and in TLR-agonist treated endothelial cells (ECs). TLR2 expression (low in control ECs) was upregulated by lipopolysaccharide, the danger signal high mobility group box-1, and hypoxia in a TLR4-dependent manner. Endothelial tube formation on Matrigel as well as EC permeability was assessed as in vitro measures of angiogenesis. Time-lapse imaging demonstrated that ECs lacking TLR4 formed more tubes, whereas TLR2 knockdown ECs exhibited attenuated tube formation. TLR2 also mediated EC permeability, an initial step during angiogenesis, in response to high-mobility group box-1 (HMGB1) that is released by cells during hypoxic injury. In vivo, ischemia-induced upregulation of TLR2 required intact TLR4 signaling that mediated systemic inflammation, as measured by local and systemic IL-6 levels. Similar to our in vitro findings, vascular density and limb perfusion were both enhanced in the absence of TLR4 signaling, but not if TLR2 was deleted. These findings indicate that TLR2, in the absence of TLR4, improves angiogenesis and perfusion recovery in response to ischemia.

MyD88 and TRIF mediate divergent inflammatory and regenerative responses to skeletal muscle ischemia.

We have previously shown that MyD88 KO mice appear protected from ischemic muscle injury while TRIF KO mice exhibit sustained necrosis after femoral artery ligation (FAL). However, our previous data did not differentiate whether the protective effect of absent MyD88 signaling was secondary to attenuated injury after FAL or quicker recovery from the insult. The purpose of this study was to delineate these different possibilities. On the basis of previous findings, we hypothesized that MyD88 signaling promotes enhanced inflammation while TRIF mediates regeneration after skeletal muscle ischemia. Our results show that after FAL, both MyD88 KO mice and TRIF KO mice have evidence of ischemia, as do their control counterparts. However, MyD88 KO mice had lower levels of serum IL-6 24 h after FAL, while TRIF KO mice demonstrated sustained serum IL-6 up to 1 week after injury. Additionally, MyD88 KO mice had higher nuclear content and larger myofibers than control animals 1 week after injury. IL-6 is known to have differential effects in myoblast function, and can inhibit proliferation and differentiation. In tibialis anterior muscle harvested from injured animals, IL-6 levels were higher and the proliferative marker MyoD was lower in TRIF KO mice by PCR. Furthermore, expression of MyD88 appeared to be higher in skeletal muscle of TRIF KO mice. In vitro, we showed that myoblast differentiation and proliferation were attenuated in response to IL-6 treatment giving credence to the finding that low IL-6 in MyD88 KO mice may be responsible for larger myocyte sizes 1 week after FAL. We conclude that MyD88 and TRIF work in concert to mediate a balanced response to ischemic injury.

HMGB1 and TLR4 mediate skeletal muscle recovery in a murine model of hindlimb ischemia.

BACKGROUND: We have previously shown that the danger signal high-mobility group box 1 (HMGB1) promotes angiogenesis when administered to ischemic muscle. HMGB1 signals through Toll-like receptor 4 (TLR4) as well as the receptor for advanced glycation end-products (RAGE). However, the actions of these receptors in ischemic injury and muscle recovery are not known. We hypothesize that TLR4 mediates tissue recovery and angiogenesis in response to ischemia. METHODS: Femoral artery ligation was performed in control, TLR4 competent (C3H/HeOuJ) and incompetent (C3H/HeJ) mice, as well as RAGE knockout mice and their C57B6 control counterparts. In other experiments, control mice were pretreated with anti-HMGB1 neutralizing antibody before femoral artery ligation. After 2 weeks, limb perfusion was evaluated using laser Doppler perfusion imaging and reported as the ratio of blood flow in the ischemic to nonischemic limb. Muscle necrosis, fat replacement, and vascular density in the anterior tibialis muscle were quantified histologically. In vitro, TLR4 and RAGE expression was evaluated in human dermal microvascular endothelial cells in response to hypoxia. Human dermal microvascular endothelial cells treated with HMGB1 alone and in the presence of anti-TLR4 antibody were probed for phosphorylated extracellular signal-regulated kinase (ERK), a signaling molecule critical to endothelial cell (EC) angiogenic behavior. RESULTS: Both anti-HMGB1 antibody as well as defective TLR4 signaling in HeJ mice resulted in prominent muscle necrosis 2 weeks after femoral artery ligation. Control HeOuJ mice had less necrosis than TLR4 incompetent HeJ mice, but a greater amount of fat replacement. In contrast to control C3H mice, control C57B6 mice demonstrated prominent muscle regeneration with very little necrosis. Muscle regeneration was not dependent on RAGE. While vascular density did not differ between strains, mice with intact RAGE and TLR4 signaling had less blood flow in ischemic limbs compared with mutant strains. In vitro, EC TLR4 expression increased in response to hypoxia while TLR4 antagonism decreased HMGB1-induced activation of extracellular signal-regulated kinase. CONCLUSIONS: Both HMGB1 and TLR4 protect against muscle necrosis after hindlimb ischemia. However, muscle regeneration does not appear to be tied to vascular density. HMGB1 likely activates angiogenic behavior in ECs in vitro, and this activation may be modulated by TLR4. The improvement in blood flow seen in mice with absent TLR4 and RAGE signaling may suggest anti-angiogenic roles for both receptors, or vasoconstriction induced by TLR4 and RAGE mediated inflammatory pathways.

TLR2 and TLR4 mediate differential responses to limb ischemia through MyD88-dependent and independent pathways.

INTRODUCTION: The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-ß (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling. METHODS: Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism. RESULTS: While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro. DISCUSSION: TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.

High mobility group box 1 promotes endothelial cell angiogenic behavior in vitro and improves muscle perfusion in vivo in response to ischemic injury.

OBJECTIVES: The angiogenic drive in skeletal muscle ischemia remains poorly understood. Innate inflammatory pathways are activated during tissue injury and repair, suggesting that this highly conserved pathway may be involved in ischemia-induced angiogenesis. We hypothesize that one of the endogenous ligands for innate immune signaling, high mobility group box 1 (HMGB1), in combination with autophagic responses to hypoxia or nutrient deprivation, plays an important role in angiogenesis. METHODS: Human dermal microvascular endothelial cells (ECs) were cultured in normoxia or hypoxia (1% oxygen). Immunocytochemical analysis of HMGB1 subcellular localization, evaluation of tube formation, and Western blot analysis of myotubule light-chain 3I (LC3I) conversion to LC3II, as a marker of autophagy, were conducted. 3-Methyladenine (3MA), chloroquine, or rapamycin were administered to inhibit or promote autophagy, respectively. In vivo, a murine hind limb ischemia model was performed. Muscle samples were collected at 4 hours to evaluate for nuclear HMGB1 and at 14 days to examine endothelial density. Perfusion recovery in the hind limbs was calculated by laser Doppler perfusion imaging (LDPI). RESULTS: Hypoxic ECs exhibited reduced nuclear HMGB1 staining compared with normoxic cells (mean fluorescence intensity, 186.9 ± 17.1 vs 236.0 ± 1.6, P = .01) with a concomitant increase in cytosolic staining. HMGB1 treatment of ECs enhanced tube formation, an angiogenic phenotype of ECs. Neutralization of endogenous HMGB1 markedly impaired tube formation and inhibited LC3II formation. Inhibition of autophagy with 3MA or chloroquine abrogated tube formation, whereas its induction with rapamycin enhanced tubing and promoted HMGB1 translocation. In vivo, ischemic skeletal muscle showed reduced numbers of HMGB1-positive myocyte nuclei compared with nonischemic muscle (34.9% ± 1.9% vs 51.7% ± 2.0%, P < .001). Injection of HMGB1 into ischemic hind limbs increased perfusion recovery by 21% and increased EC density (49.2 ± 4.1 vs 34.2 ± 3.4 ECs/high-powered field, respectively; P = .02) at 14 days compared with control hind limbs. CONCLUSIONS: Nuclear release of HMGB1 and autophagy occur in ECs in response to hypoxia or serum depletion. HMGB1 and autophagy are necessary and likely play an interdependent role in promoting the angiogenic behavior of ECs. In vivo, HMGB1 promotes perfusion recovery and increased EC density after ischemic injury. These findings suggest a possible mechanistic link between autophagy and HMGB1 in EC angiogenic behavior and support the importance of innate immune pathways in angiogenesis.

The search for a useful method for the optimal cryopreservation of adipose aspirates: part II. In vivo study.

PURPOSE: The previous in vitro study showed that trehalose, when used as a cryoprotective agent (CPA) in an optimal concentration, can provide adequate protection of adipose aspirates during cryopreservation. OBJECTIVE: The authors evaluated the efficacy of trehalose in its optimal concentration for cryopreservation of human fat grafts in a well-established animal model. METHODS: In this study (n = 20 in each group), adipose aspirates were harvested and processed from a female patient; the protocol for freezing and thawing of fat grafts was the same as the in vitro study. In the control group, 0.5 mL of fresh fat grafts was injected into the posterior scalp of a nude mouse. In the cryopreservation group 1, a combination of dimethyl sulfoxide (in 0.5M) and trehalose (in 0.2M) was injected as a CPA. In the cryopreservation group 2, only the optimal concentration of trehalose (in 0.35M) was administered as a CPA. In both cryopreservation groups, 0.5 mL of cryopreserved fat grafts was thawed and injected into the animal in the same manner as the control group. All animals in each group were observed for gross appearance of maintained fat grafts over their posterior scalps for up to eight weeks. The final volume and weight of maintained fat grafts and their histology were evaluated at the end of the study. RESULTS: Group 2, compared with group 1, respectively, had equivalently maintained volume (38.2 +/- 10.1% versus 46.1 +/- 14.4%, ns) and weight (34.1 +/- 12.1% versus 38.9 +/- 14.7%, ns). However, the results from both cryopreservation groups were still inferior to those from the control group (both P < .05). Histologically, the basic structure of adipose tissue was maintained in all three groups. CONCLUSION: Trehalose, serving as a CPA in its optimal concentration, appears to provide adequate protection of human fat grafts during cryopreservation in vivo. Such protection is similar to that provided by the combination of dimethyl sulfoxide and trehalose as a CPA. Because of its safety and effectiveness, trehalose can possibly be administered to patients for long-term preservation of their fat grafts.

Cryopreservation of autologous fat grafts harvested with the Coleman technique.

The viability of fat grafts harvested with an established technique after cryopreservation remains unknown. This study was conducted in vitro to evaluate the viability of autologous fat grafts harvested with the Coleman technique and subsequently preserved with our preferred cryopreservation method. Eight adult females were enrolled in this study. In each patient, 10 mL of fat grafts were harvested with the Coleman technique by a single surgeon from the lower abdomen. In group 1, 5 mL of fresh fat grafts were mixed with cryoprotective agents and underwent cryopreservation with controlled slow cooling and fast rewarming. In group 2, 5 mL of fresh fat grafts without cryopreservation from the same patient served as a control. The fat graft samples from both groups were evaluated with trypan blue vital staining, glycerol-3-phophatase dehydrogenase assay, and routine histology. Viable adipocyte counts were found similar in both group 1 and group 2 (3.46 +/- 0.91 vs. 4.12 +/- 1.11 x 10/mL, P = 0.22). However, glycerol-3-phophatase dehydrogenase activity was significantly lower in group 1 compared with group 2 (0.47 +/- 0.09 vs. 0.66 +/- 0.09 u/mL, P < 0.001). Histologically, the normal structure of fragmented fatty tissues was found primarily in both groups. Our results indicate that autologous fat grafts harvested with the Coleman technique and preserved with our preferred cryopreservation method have a normal histology with near the same number of viable adipocytes as compared with the fresh fat grafts. However, those cryopreserved fat grafts appear to have a less optimal level of adipocyte specific enzyme activity compared with the fresh ones and thus may not survive well after they are transplanted without being optimized.

The search for a useful method for the optimal cryopreservation of adipose aspirates: part I. In vitro study.

BACKGROUND: Previous studies have shown that trehalose, when used alone as a cryoprotective agent (CPA), can maintain the viability of living tissue after cryopreservation and therefore may be used clinically for the future banking of human fat grafts. OBJECTIVE: The purpose of this study is to determine the optimal concentration of trehalose used as a CPA for the cryopreservation of adipose aspirates in vitro. METHODS: Adipose tissue was harvested with conventional liposuction from the abdomens of eight female patients and adipose aspirates were then collected from the resulting middle layer after centrifugation. A 3-cc specimen of adipose aspirates was cryopreserved using trehalose as a CPA in seven different concentrations (0.20, 0.25, 0.30, 0.35, 0.40, 0.50, and 0.75 mol/L, respectively). Cryopreservation was conducted with controlled slow cooling to -196 degrees C and fast re-warming to 37 degrees C, according to our established protocol. A 3-cc specimen of fresh adipose aspirates without cryopreservation served as the control. Fresh or cryopreserved adipose aspirates in each group were evaluated by viable adipocyte counts, glycerol-3-phosphate dehydrogenase (G3PDH) assay, and histology. RESULTS: More viable adipocytes of cryopreserved adipose aspirates were found when a 0.35 mol/L concentration of trehalose was used, as compared to the other cryopreserved groups. In terms of viable adipocyte counts, there was no statistical difference between the fresh control group and the cryopreserved group with trehalose in that concentration (1.88 +/- 0.61 vs. 2.4 +/- 0.52 x 10(6)/mL; P > .05). G3PDH assay was performed to assess intracellular function and showed no statistical significance in the fresh control group compared with all cryopreserved groups (all P > .05). Histologically, the basic structure of adipose tissue was adequately maintained in most of the cryopreserved groups. CONCLUSIONS: Trehalose as a CPA with a concentration of 0.35 mol/L appears to provide the optimal protection of adipose aspirates during cryopreservation. Further in vivo study will be needed to confirm these findings.

Autologous fat grafts harvested and refined by the Coleman technique: a comparative study.

BACKGROUND: The viability of fat grafts obtained by even a well-established technique remains poorly studied and unknown. This study was designed to determine the viability of fat grafts harvested and refined by the Coleman technique. METHODS: Sixteen adult white women were enrolled in this study. In group 1 (n = 8), fat grafts were harvested and processed with the Coleman technique by a single surgeon from the abdomen of each patient according to his standardized method. In group 2 (n = 8), fat grafts were harvested with the conventional liposuction by another surgeon. After centrifugation, the resulting middle layer of tissue was collected. All fat graft samples were analyzed for the following studies: trypan blue vital staining for viable adipocyte counts, glycerol-3-phophatase dehydrogenase assay, and routine histologic examination. RESULTS: The higher viable adipocyte counts were found in group 1 compared with group 2 (4.11 +/- 1.11 versus 2.57 +/- 0.56 x 10 cells/ml; p < 0.004). The level of glycerol-3-phophatase dehydrogenase activity was significantly higher in group 1 compared with group 2 (0.66 +/- 0.09 versus 0.34 +/- 0.13 U/ml; p < 0.0001). Histologic examination showed normal structure of fragmented fatty tissues in both groups. CONCLUSIONS: Although fat grafts obtained by both methods maintain normal histologic structure, the Coleman technique yields a greater number of viable adipocytes and sustains a more optimal level of cellular function within fat grafts and should be considered superior to conventional liposuction as a preferred method of choice for fat graft harvesting.

Cryopreservation of composite tissue transplants.

Composite tissue allotransplantation holds great promise for upper extremity reconstruction but is limited by donor part availability. Cryopreservation may increase the availability of donor parts and even reduce antigenicity. The purpose of the study was to evaluate the viability of cryopreserved composite tissues and to demonstrate the feasibility of microvascular isotransplantation of cryopreserved composite flaps. Twenty epigastric flaps were harvested from Lewis rats. Ten flaps were analyzed fresh. Ten flaps were perfused with dimethyl sulfoxide (DMSO)/trehelose cryoprotectant agent (CPA), frozen by controlled cooling to -140 degrees C, and stored for 2 weeks. Flaps were evaluated by factor VIII endothelial staining and MTT tetrazolium salt assay. For the in vivo phase, 30 flaps were harvested. Ten were transplanted fresh to isogenetic recipient animals, ten were perfused with CPA and transplanted, and ten were cryopreserved for 2 weeks, thawed, and transplanted. All cryopreserved samples displayed intact vascular endothelia on factor VIII staining. On MTT analysis, the epithelial viability index for the cryopreserved samples was not significantly different from fresh controls (p = 0.12). All freshly transplanted flaps (10/10) were viable at 60 days. Nine of ten flaps in the perfused/transplanted group were viable at 60 days. Survival of cryopreserved/transplanted flaps ranged from 5 to 60 days. The skin and vascular endothelial components of composite tissue flaps appear to retain their viability after cryopreservation. The in vivo studies demonstrate that the long-term survival of cryopreserved composite tissue transplants is feasible and support an indirect injury, rather than direct injury from freezing or cryoprotectant agents, as the mechanism of flap loss.

The viability of autologous fat grafts harvested with the LipiVage system: a comparative study.

This study evaluates the viability of adipose aspirates harvested with the LipiVage system (Genesis Biosystems Inc, Lewisville, TX), a newly developed fat harvesting device, and determines a potentially preferred method for possible large-quantity fat graft harvesting. Adipose aspirates were harvested with the LipiVage system from the abdomen of 16 female patients (group 1, n = 8) according to the instruction by the manufacturer and with conventional liposuction (group 2, n = 8). Samples from conventional liposuction were spun at 50 g for 10 minutes and the resulting middle layer of fat was collected. All fat graft samples were evaluated with trypan blue vital staining for viable adipocyte count, glycerol-3-phosphatase dehydrogenase (G3PDH) assay for intracellular enzyme activity, and histology. In this study, group 1 had significantly higher viable adipocyte count than group 2 had (3.7 +/- 0.64 versus 2.37 +/- 0.56 x 10(6) /mL, P = 0.0021). G3PDH assay showed a marked increase of intracellular enzyme activity in group 1 compared with in group 2 (0.61 +/- 0.10 versus 0.34 +/- 0.13 U/mL, P = 0.00045). Histology revealed normal structures of fragmental fatty tissues in both groups. While adipose aspirates by both modalities maintain normal structure, the LipiVage system yields a greater number of viable adipocytes and sustains a higher level of intracellular enzyme activity within fat grafts and can potentially be a preferred method of choice for large-quantity fat graft harvesting.

Cryopreservation of composite tissues and transplantation: preliminary studies.

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5M dimethyl sulfoxide (Me(2)SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to -140 degrees C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n=10), perfused (n=8), and cryopreserved (n=17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n=10) was 11.17+/-2.01, which was not significantly different from fresh controls (n=10, 12.15+/-1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.

Cryopreservation of composite tissue flaps: experimental studies in the rat.

Cryopreservation has the potential to improve availability of donor parts for composite tissue allotransplantation and may reduce their antigenicity. This study investigates whether the component tissues of composite flaps remain viable after cryopreservation. Forty-one epigastric flaps were harvested from Lewis rats. Twenty-one flaps were perfused with DMSO/trehalose, frozen by controlled cooling to -140 degrees C, and stored in liquid nitrogen for 2 weeks. Ten fresh and 10 cryopreserved/thawed flaps were examined histologically with hematoxylin & eosin and factor VIII staining. An epithelial viability index was calculated for 10 fresh and 11 cryopreserved flaps using the MTT assay. In all cryopreserved samples, hematoxylin & eosin, and factor VIII staining revealed a well-preserved cellular architecture, which was indistinguishable from fresh specimens. The viability index for the cryopreserved samples was 10.90 +/- 2.09 compared with 12.15 +/- 1.32 for fresh flaps (P = 0.123). Results suggest that the skin, adipose, and vascular endothelial cells of composite tissue flaps retain their viability after cryopreservation and thawing.

Adipose aspirates as a source for human processed lipoaspirate cells after optimal cryopreservation.

BACKGROUND: The purpose of this study was to test the authors' hypothesis that previously cryopreserved adipose aspirates collected from conventional liposuction could still be a reliable source of human processed lipoaspirate cells. METHODS: Adipose aspirates were collected from 12 adult female patients after conventional liposuction of the abdomen and were then preserved by an optimal cryopreservation method with added cryoprotective agents (0.5 M dimethyl sulfoxide and 0.2 M trehalose). Cryopreservation of the adipose tissues was subsequently conducted with controlled slow cooling and then stored in liquid nitrogen (-196 degrees C). One gram of fresh or cryopreserved (after fast rewarming) adipose aspirates was processed in vitro and the resulting cell pellet, consisting of processed lipoaspirate cells, was cultured separately. The length of time until processed lipoaspirate cells became adherent to the culture plate was recorded and the number of processed lipoaspirate cells after a 2-week culture was counted. RESULTS: Flat, spindle-shape processed lipoaspirate cells from the cryopreserved group became adherent to the plate within 48 to 72 hours after initial culture compared with the fresh group, where the cells became adherent by 24 hours. After a 2-week culture, the cryopreserved aspirates yielded an average of 3.7 +/- 1.4 x 10(5) processed lipoaspirate cells per milliliter, equal to 90 percent of the yielded number of cells obtained from the fresh aspirates (4.1 +/- 1.4 x 10(5) cells/ml). CONCLUSIONS: The authors' results indicate that although there is a latency of cell growth after an optimal cryopreservation, cryopreserved adipose aspirates can yield a significant number of processed lipoaspirate cells compared with fresh aspirates and may be a reliable source of human processed lipoaspirate cells because they can still be processed later after long-term preservation.