Treatment of chronic diabetic foot ulcers with adipose-derived stromal vascular fraction cell injections: Safety and evidence of efficacy at 1 year.

Diabetes affects multiple systems in complex manners. Diabetic foot ulcers (DFUs) are a result of diabetes-induced microarterial vessel disease and peripheral neuropathy. The presence of arteriosclerosis-induced macroarterial disease can further complicate DFU pathophysiology. Recent studies suggest that mesenchymal stromal cell therapies can enhance tissue regeneration. This phase I study was designed to determine the safety and explore the efficacy of local injections of autologous adipose-derived stromal vascular fraction (SVF) cells to treat nonhealing DFUs greater than 3 cm in diameter. Sixty-three patients with type 2 diabetes with chronic DFU-all amputation candidates-were treated with 30 × 106 SVF cells injected in the ulcer bed and periphery and along the pedal arteries. Patients were seen at 6 and 12 months to evaluate ulcer closure. Doppler ultrasounds were performed in a subset of subjects to determine vascular structural parameters. No intervention-related serious adverse events were reported. At 6 months, 51 subjects had 100% DFU closure, and 8 subjects had ≥75% closure. Three subjects had early amputations, and one subject died. At 12 months, 50 subjects had 100% DFU healing and 4 subjects had ≥85% healing. Five subjects died between the 6- and 12-month follow-up visits. No deaths were intervention related. Doppler studies in 11 subjects revealed increases in peak systolic velocity and pulsatility index in 33 of 33 arteries, consistent with enhanced distal arterial runoff. These results indicate that SVF can be safely used to treat chronic DFU, with evidence of efficacy (wound healing) and mechanisms of action that include vascular repair and/or angiogenesis.

Sustained clinical improvement of Parkinson's disease in two patients with facially-transplanted adipose-derived stromal vascular fraction cells.

Cell-based therapy has been studied as an alternative for Parkinson's Disease (PD), with different routes of administration. The superficial fascia and facial muscles possess a rich blood supply, while venous and lymphatic access via the orbit and the cribriform plate provide a route to cerebral circulation. We here document positive clinical effects in two patients with PD treated with autologous adipose-derived stromal vascular fraction (SVF) cell preparation, implanted into the face and nasal cavity. Two patients with PD were transplanted with 60 million total nucleated cells in processed SVF into the facial muscles and nose. Serial evaluations were carried out up to 5 years (patient 1) and 1 year (patient 2), using the PDQ-39, the UPDRS, and serial videos. Video scoring was reviewed in a blinded fashion. Both patients reported qualitative improvement in motor and nonmotor symptoms following injection. Quantitatively, PDQ-39 scores decreased in all categories for both. On-medication UPDRS motor scores decreased in both (20 to 4 in patient 1, 18 to 3 in patient 2) despite taking the same or less medication (LEDD 350 to 350 in patient 1, LEDD 1175 to 400 in pt2). Both subjects had off-medication UPDRS scores similar to their pretreatment on-medication scores (20 to 14 in patient 1, 18 to 23 in patient 2). These preliminary findings describe local facial and nasal injections of SVF preparation followed by prolonged clinical benefit in two patients. Despite an unknown mechanism of action, this potential therapy warrants careful verification and investigation.

Intra-articular infiltration of adipose-derived stromal vascular fraction cells slows the clinical progression of moderate-severe knee osteoarthritis: hypothesis on the regulatory role of intra-articular adipose tissue.

BACKGROUND: The infiltration of the stromal vascular fraction (SVF) of autologous adipose tissue to treat osteoarthritis has been used for several years demonstrating its safety and noticeable efficacy. This article presents clinical data from patients afftected by moderate and severe knee osteoarthritis demonstrating safety and clinical efficacy of the treatment when this autologous cell product is injected in the knee joint and patients evaluated post-operatively after 1 year. However, what do we know about the mechanism that underlies this clinical improvement? This article proposes, for the first time in our opinion, a hypothesis of the mode of action that involves structural and molecular interactions between SVF and infrapatellar fat pad (IFP). As consequence, there would be a re-education of intra-articular adipose tissue, which we consider a key player for the clinical effect observed in the mid and long term mainly due to immuno-regulatory mechanisms. METHODS: This is a retrospective and not controlled study that evaluated 50 patients (100 joints) ranging from 50 to 89 years old, separated by age cohorts. Clinical efficacy was assessed using the Lequesne, WOMAC, and VAS scales, by ultrasound control and quantification of the biochemical profiles of synovial fluid. RESULTS: There were no serious adverse effects. All the indexes studied showed a significant clinical improvement after 1-year follow-up for all ages and OA degree groups. This finding was correlated with the ultrasound observations and biochemical data, which show a marked decrease in catabolic and pro-inflammatory molecules (MMP-2, IL-1B, IL-6, and IL-8) and significant increase for anabolic and anti-inflammatory molecules (IGF-1 and IL-10). CONCLUSIONS: We conclude that intra-articular SVF infiltration for knee OA treatment is safe and effective during 1 year. We propose that applied SVF cells cause a cascade of molecular and structural events that, through complex interactions between IFP and SVF, re-educating the intra-articular fatty tissue towards a homeostatic, protective, and anti-inflammatory function, which will ultimately promote the restructuring and regeneration of damaged tissues.

Reproducible Volume Restoration and Efficient Long-term Volume Retention after Point-of-care Standardized Cell-enhanced Fat Grafting in Breast Surgery.

UNLABELLED: Lipoaspirated fat grafts are used to reconstruct volume defects in breast surgery. Although intraoperative treatment decisions are influenced by volume changes observed immediately after grafting, clinical effect and patient satisfaction are dependent on volume retention over time. The study objectives were to determine how immediate breast volume changes correlate to implanted graft volumes, to understand long-term adipose graft volume changes, and to study the "dose" effect of adding autologous stromal vascular fraction (SVF) cells to fat grafts on long-term volume retention. METHODS: A total of 74 patients underwent 77 cell-enhanced fat grafting procedures to restore breast volume deficits associated with cosmetic and reconstructive indications. Although all procedures used standardized fat grafts, 21 of the fat grafts were enriched with a low dose of SVF cells and 56 were enriched with a high SVF cell dose. Three-dimensional imaging was used to quantify volume retention over time. RESULTS: For each milliliter of injected fat graft, immediate changes in breast volume were shown to be lower than the actual volume implanted for all methods and clinical indications treated. Long-term breast volume changes stabilize by 90-120 days after grafting. Final volume retention in the long-term was higher with high cell-enhanced fat grafts. CONCLUSIONS: Intraoperative immediate breast volume changes do not correspond with implanted fat graft volumes. In the early postoperative period (7-21 days), breast volume increases more than the implanted volume and then rapidly decreases in the subsequent 30-60 days. High-dose cell-enhanced fat grafts decrease early postsurgical breast edema and significantly improve long-term volume retention.

Signaling pathways involved in antidepressant-induced cell proliferation and synaptic plasticity.

In the last years it has been proposed that the antidepressant action is mediated not only by changes in monoamine levels but also in association with modifications involving cell proliferation and plasticity in some brain limbic areas as hippocampus, and also frontal cortex and amygdala. This leads to the merging of the classic "monoaminergic hypothesis of depression", with the newer "neurotrophic hypothesis of depression". Here we review two important signaling pathways: the Wnt/ß-catenin pathway -implicated in cellular proliferation and synaptic plasticity- that is downregulated in major depression and upregulated after antidepressant treatment; and the mTOR pathway -controling synaptic plasticity- recently related to present disrupted functioning in major depression, and as the target of some drugs with fast-acting potential antidepressant action. These pieces of evidences are confirmed in a variety of animal models of depression and are predictive of antidepressant actions. We also review the role of another two important neurotrophic factors: brain derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) that mediate the antidepressant effects. All of the above intracellular pathways interact by a crosstalk mediated by Akt, a key regulator molecule that may underlie the fine tuning between proliferative and neuroplasticity changes induced by antidepressant drugs.

GABA(A) receptor chloride channels are involved in the neuroprotective role of GABA following oxygen and glucose deprivation in the rat cerebral cortex but not in the hippocampus.

Assays on "ex vivo" sections of rat hippocampus and rat cerebral cortex, subjected to oxygen and glucose deprivation (OGD) and a three-hour reperfusion-like (RL) recovery, were performed in the presence of either GABA or the GABA(A) receptor binding site antagonist, bicuculline. Lactate dehydrogenase (LDH) and propidium iodide were used to quantify cell mortality. We also measured, using real-time quantitative polymerase chain reaction (qPCR), the early transcriptional response of a number of genes of the glutamatergic and GABAergic systems. Specifically, glial pre- and post-synaptic glutamatergic transporters (namely GLAST1a, EAAC-1, GLT-1 and VGLUT1), three GABAA receptor subunits (α1, ß2 and γ2), and the GABAergic presynaptic marker, glutamic acid decarboxylase (GAD65), were studied. Mortality assays revealed that GABAA receptor chloride channels play an important role in the neuroprotective effect of GABA in the cerebral cortex, but have a much smaller effect in the hippocampus. We also found that GABA reverses the OGD-dependent decrease in GABA(A) receptor transcript levels, as well as mRNA levels of the membrane and vesicular glutamate transporter genes. Based on the markers used, we conclude that OGD results in differential responses in the GABAergic presynaptic and postsynaptic systems.

Neural plasticity and proliferation in the generation of antidepressant effects: hippocampal implication.

It is widely accepted that changes underlying depression and antidepressant-like effects involve not only alterations in the levels of neurotransmitters as monoamines and their receptors in the brain, but also structural and functional changes far beyond. During the last two decades, emerging theories are providing new explanations about the neurobiology of depression and the mechanism of action of antidepressant strategies based on cellular changes at the CNS level. The neurotrophic/plasticity hypothesis of depression, proposed more than a decade ago, is now supported by multiple basic and clinical studies focused on the role of intracellular-signalling cascades that govern neural proliferation and plasticity. Herein, we review the state-of-the-art of the changes in these signalling pathways which appear to underlie both depressive disorders and antidepressant actions. We will especially focus on the hippocampal cellularity and plasticity modulation by serotonin, trophic factors as brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF) through intracellular signalling pathways-cAMP, Wnt/ ß -catenin, and mTOR. Connecting the classic monoaminergic hypothesis with proliferation/neuroplasticity-related evidence is an appealing and comprehensive attempt for improving our knowledge about the neurobiological events leading to depression and associated to antidepressant therapies.

New strategies in the development of antidepressants: towards the modulation of neuroplasticity pathways.

Over the past five decades, the pharmacological treatment of depression has been based on the pathophysiological hypothesis of a deficiency in monoamines, mainly serotonin and noradrenaline. Antidepressant prescribed today, all of them designed to enhance central monoaminergic tone, present several important limitations, including a 2-5 weeks response lag and also a limited clinical efficacy. As it is increasingly evident that the abnormalities associated to depression go beyond monoamines, the development of better antidepressants will depend on the identification and understanding of new cellular targets. In this regard, much evidence supports a role for cellular and molecular mechanisms of neuroplasticity, including neurotrophic inputs, in mood disorders, in parallel with the biological features associated to stress conditions. In order to illustrate the possible relevance of neuroplasticity-related pathways for the therapy of depressive states, we here review the biological evidence supporting some therapeutic strategies in a very initial phase of development (modulation of the Wnt/GSK-3ß/ß-catenin pathway, potentiation of endocannabinoid activity, agonism of 5-HT(4) receptors), which involve modulation of downstream mechanisms and neuroplasticity circuits. These strategies also show the existence of mixed mechanisms of action, constituting a nexus between the "classic" aminergic theory and the "new" neuroplasticity hypothesis.

Age-dependent modifications in the mRNA levels of the rat excitatory amino acid transporters (EAATs) at 48hour reperfusion following global ischemia.

This study reports the mRNA levels of some excitatory amino acid transporters (EAATs) in response to ischemia-reperfusion (I/R) in rat hippocampus and cerebral cortex. The study was performed in 3-month-old and 18-month-old animals to analyze the possible role of age in the I/R response of these transporters. The I/R resulted in a reduced transcription of both the neuronal EAAC1 (excitatory amino acid carrier-1) and the neuronal and glial GLT-1 (glial glutamate transporter 1), while the glial GLAST1a (l-glutamate/l-aspartate transporter 1a) transcription increased following I/R. The changes observed were more striking in 3-month-old animals than in 18-month-old animals. We hypothesize that increases in the GLAST1a mRNA levels following I/R insult can be explained by increases in glial cells, while the GLT-1 response to I/R mirrors neuronal changes. GLAST1a transcription increases in 3-month-old animals support the hypothesis that this transporter would be the main mechanism for extracellular glutamate clearance after I/R. Decreases in EAAC1 and GLT-1 mRNA levels would represent either neuronal changes due to the delayed neuronal death or a putative protective down-regulation of these transporters to decrease the amount of glutamate inside the neurons, which would decrease their glutamate release. This study also reports how the treatment with the anti-inflammatory agent meloxicam attenuates the transcriptional response to I/R in 3-month-old rats and decreases the survival of the I/R-injured animals.

Age and meloxicam attenuate the ischemia/reperfusion-induced down-regulation in the NMDA receptor genes.

This study describes the effect of global brain ischemia followed by 48 h reperfusion, when delayed neuronal death can be already observed. We quantified the mRNA levels of the N-methyl-D-aspartate receptor (NMDAR) subunits and those of the astroglia (glial fibrilar acidic protein, GFAP) and microglia (CD11b) markers using real time PCR on the cerebral cortex and hippocampus of 3- and 18-month-old Sprague-Dawley rats. Data show an ischemia/reperfusion-induced decrease in the mRNA levels of the NMDAR NR1, NR2A and NR2B subunits genes, which contrasts with the increase in the CD11b and GFAP mRNA levels. These effects are attenuated in all the genes studied in 18-month-old animals, suggesting that this mechanism of response is less efficient in aged animals. Western blot assays of NR1, NR2A and NR2B show parallels with the real time PCR data, indicating that the down-regulation of these genes is controlled at the transcriptional level. We suggest that a decrease in the efficiency in the control of the NMDAR transcription could account for the higher vulnerability in aged animals, but it cannot explain by itself differences in the vulnerability to ischemia in different areas of the brain. In the assays of ischemia/reperfusion followed by a treatment with the anti-inflammatory agent meloxicam, we observed that ischemic insult was unable to elicit changes in the NMDAR transcription, thus suggesting that inflammation plays a crucial role in the transcriptional control of these genes.

Functional autoradiography and gene expression analysis applied to the characterization of the alpha2-adrenergic system in the chicken brain.

Here we report a functional autoradiographic study of [(35)S]GTPgammaS binding induced by alpha(2)-adrenoceptor activation in chicken brain tissue sections using both 10(-4)M UK 14304 (bromoxidine or brimonidine) and 10(-6)M epinephrine as alpha(2)-adrenoceptor agonists. Assays were performed using two different incubation buffers: glycylglycine or Tris-HCl. Changes in the [(35)S]GTPgammaS basal binding values were detected, and different [(35)S]GTPgammaS specific binding values were also obtained depending on the buffer used for each drug. The best results were obtained with epinephrine in Tris-HCl, with slightly higher stimulation values than the observed with UK 14304 in glycylglycine buffer. The effect of the addition of adenosine deaminase to the incubation buffer was also tested. This effect decreasing basal binding in chicken was very small when compared to mammals, according with differences found in adenosine 1 receptor expression levels. Structures presenting alpha(2)-adrenoceptor-mediated G(i/o) protein stimulation fitted with areas previously described as enriched in alpha(2)-adrenoceptors in chicken brain, and their homologous areas in mammals. These data confirm the specificity of the results and reinforce the implication of the alpha(2)-adrenoceptors in the function of these brain nuclei. On the other hand, the expression level of the different alpha(2)-adrenoceptor subtypes was tested with real-time PCR. Contrasting with the alpha(2)-adrenoceptor subtype distribution previously described with radioligand competition assays, where alpha(2A) was the predominant alpha(2)-adrenoceptor subtype (>/=75%); in the present work, the ratio of alpha(2A):alpha(2B/C) gene expression was lower than expected both in telencephalon, tectum opticum, and cerebellum.

Global ischemia-induced modifications in the expression of AMPA receptors and inflammation in rat brain.

Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPAR) and inflammatory processes have been related to ischemia-induced damage, but there are few studies addressing their response in different brain areas. Here we compare AMPAR expression after ischemia in several brain areas (hippocampus, cerebral cortex and caudate-putamen) in an attempt to correlate it with their different vulnerabilities. We found outstanding decreases in GluR1 and GluR2 mRNA levels after global ischemia and 48 h reperfusion (I/R) in all the areas studied, however, protein levels maintained in some areas such as CA3, suggesting different post-transcriptional control in different areas of the brain. To characterize the inflammatory response in these areas, we measured the mRNA levels of CD11b/CD18 membrane integrin (a reactive microglia marker), which showed an important but similar up-regulation in all brain areas studied, which was confirmed by immunohistochemistry. We conclude that the down-regulation of AMPAR gene expression following I/R does not explain differences in the vulnerability of different areas. Additionally, our data indicate that the level of inflammation is independent of the vulnerability of the different brain areas and does not explain differences in the AMPAR expression observed in the brain areas studied.

Transient global ischemia in rat brain promotes different NMDA receptor regulation depending on the brain structure studied.

The mRNA expression of the major subunits of N-methyl-d-aspartate receptors (NR1, NR2A and NR2B) following ischemia-reperfusion was studied in structures with different vulnerabilities to ischemic insult in the rat brain. The study was performed using quantitative real-time PCR on samples from 3-month-old male Sprague-Dawley rats after global transient forebrain ischemia followed by 48h of reperfusion. Expression of NMDA receptor subunits mRNAs decreased significantly in all structures studied in the injured animals as compared to the sham-operated ones. The hippocampal subfields (CA1, CA3 and dentate gyrus) as well as the caudate-putamen, both reported to be highly ischemic-vulnerable structures, showed outstandingly lower mRNA levels of NMDA receptor subunits than the cerebral cortex, which is considered a more ischemic-resistant structure. The ratios of the mRNA levels of the different subunits were analyzed as a measure of the NMDA receptor expression pattern for each structure studied. Hippocampal areas showed changes in NMDA receptor expression after the insult, with significant decreases in the NR2A with respect to the NR1 and NR2B subunits. Thus, the NR1:NR2A:NR2B (1:1:2) ratios observed in the sham-operated animals became (2:1:4) in insulted animals. This modified expression pattern was similar in CA1, CA3 and the dentate gyrus, in spite of the different vulnerabilities reported for these hippocampal areas. In contrast, no significant differences in the expression pattern were observed in the caudate-putamen or cerebral cortex on comparing the sham-operated animals with the ischemia-reperfused rats. Our results support the notion that the regulation of NMDA receptor gene expression is dependent on the brain structure rather than on the higher or lower vulnerability of the area studied.

Muscarinic receptor changes in the gerbil thalamus during aging.

Here we studied muscarinic receptors in the gerbil thalamus at 8 different ages - from 6 to 36 months - using receptor and functional autoradiography. The pharmacological profile inhibiting [(3)H]N-methyl scopolamine ([(3)H]NMS) binding with 50 and 200 nM pirenzepine, 30 nM pFHHSiD and 100 nM AF-DX 116 revealed the predominance of the M(2) muscarinic subtype in the thalamic nuclei studied, mainly in the anteroventral, anteromedial and paraventricular thalamic nuclei. These data correlated with the highest [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTP gamma S) binding induced in these nuclei by the muscarinic agonist oxotremorine in functional autoradiographic assays. Significant aging-dependent increases in the functional response in these three nuclei were observed, but only the anteroventral and anteromedial thalamic nuclei showed aging-dependent increases in [(3)H]NMS binding. Since these nuclei exert relevant functions, in which cholinergic pathways are involved and acetylcholine release is reported to decrease during aging, we suggest that the anteroventral and anteromedial thalamic nuclei would play critical roles in the cholinergic transmission that require compensatory mechanisms during the aging process and that are not observed in other thalamic nuclei.

Quantitative gene expression analysis in a brain slice model: influence of temperature and incubation media.

We describe the RNA integrity (28S/18S ratio) and the messenger RNA (mRNA) expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH), microtubule-associated serine/threonine kinase 2 (Mast2), and beta-actin in cortical brain slices incubated for up to 24h in Ringer's solution and Dulbecco's modified Eagle's medium (DMEM) at 25 and 37 degrees C. Our data reveal an optimal temporal working window between 1 and 6h when slices are incubated in Ringer's solution at 25 degrees C that allows experiments related to gene expression dynamics to be performed more suitably than those carried out at 37 degrees C. In addition, we show that reference gene expression may be modified in dynamic experiments and may compromise studies of gene expression.