Shelley J. Williams

Improved survival with early fluid resuscitation following hemorrhagic shock.

Abstract. Recent studies have questioned the benefits of early fluid resuscitation in hemorrhagic shock. The purpose of the current study is to evaluate the effects of early fluid resuscitation (HSE) (15 minutes), delayed fluid resuscitation (HSD) (60 minutes), and no fluid resuscitation (HSU) on cytokine levels, hepatic resting membrane potential (Em), renal function, and mortality. Eighty male Sprague-Dawley rats (350–450 g) were hemorrhaged 35% of their total blood volume and then received 40, 80, or 100 ml of crystalloid per kilogram as intravenous fluids (IVFs). The implementation of HSE resulted in stabilization of the Em (−29 mV), which was significantly different from that seen with HSD or HSU (−24 and −29 mV, respectively). The timing of resuscitation did not affect the elevation of tumor necrosis factor (TNFα) levels. The interleukin-6 (IL-6) levels for the HSE group were 81, 101, and 274 pg/ml for 40, 80, and 100 ml/kg, respectively. In contrast, HSD group IL-6 levels were 440, 566, and 632 pg/ml for 40, 80, and 100 ml/kg (p < 0.0001). IL-6 levels for the HSU group was 427 pg/ml, which was significantly different from that of the HSE group (p < 0.05). Urine output was present in 58% of the HSE rats but only 24% in the HSD rats and 0% of the HSU rats. Mortality was 11% for HSE, 58% for HSD, and 50% for HSU rats. Despite the recent studies questioning the benefits of early fluid resuscitation, these data show marked improvement in hepatic stability, the presence of urine output, decreased IL-6 levels, and significantly lower mortality when IVFs were given early after hemorrhagic shock. Furthermore, excessive fluid resuscitation (100 ml/kg) resulted in an increased inflammatory cytokine level and mortality and may account for the controversy.

Analysis for apoptosis and necrosis on adipocytes, stromal vascular fraction, and adipose-derived stem cells in human lipoaspirates after liposuction.

Background: Adipose-derived stem cells have become the most studied adult stem cells. The authors examined the apoptosis and necrosis rates for adipocyte, stromal vascular fraction, and adipose-derived stem cells in fresh human lipoaspirates. Methods: Human lipoaspirate (n = 8) was harvested using a standard liposuction technique. Stromal vascular fraction cells were separated from adipocytes and cultured to obtain purified adipose-derived stem cells. A panel of stem cell markers was used to identify the surface phenotypes of cultured adipose-derived stem cells. Three distinct stem cell subpopulations (CD90+/CD45−, CD105+/CD45−, and CD34+/CD31−) were selected from the stromal vascular fraction. Apoptosis and necrosis were determined by annexin V/propidium iodide assay and analyzed by flow cytometry. Results: The cultured adipose-derived stem cells demonstrated long-term proliferation and differentiation evidenced by cell doubling time and positive staining with oil red O and alkaline phosphatase. Isolated from lipoaspirates, adipocytes exhibited 19.7 ± 3.7 percent apoptosis and 1.1 ± 0.3 percent necrosis; stromal vascular fraction cells revealed 22.0 ± 6.3 percent of apoptosis and 11.2 ± 1.9 percent of necrosis; stromal vascular fraction cells had a higher rate of necrosis than adipocytes (p < 0.05). Among the stromal vascular fraction cells, 51.1 ± 3.7 percent expressed CD90+/CD45−, 7.5 ± 1.0 percent expressed CD105+/CD45−, and 26.4 ± 3.8 percent expressed CD34+/CD31−. CD34+/CD31− adipose-derived stem cells had lower rates of apoptosis and necrosis compared with CD105+/CD45− adipose-derived stem cells (p < 0.05). Conclusions: Adipose-derived stem cells had a higher rate of apoptosis and necrosis than adipocytes. However, the extent of apoptosis and necrosis was significantly different among adipose-derived stem cell subpopulations.

Nitrite attenuates ischemia-reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle.

Background: Recently, nitrite has been rediscovered as a physiologically relevant storage reservoir of nitric oxide in blood and it can readily be converted to nitric oxide under hypoxic and acidic conditions. In this study, the authors evaluated the therapeutic efficacy of nitrite on reperfusion-induced microcirculatory alterations and mitochondrial dysfunction in the microvasculature of skeletal muscle. Methods: The authors used a vascular pedicle isolated rat cremaster model that underwent 4 hours of warm ischemia followed by 2 hours or 17 hours of reperfusion. At 5 minutes before reperfusion, normal saline, sodium nitrite (0.20 &mgr;M/minute/kg), or nitrite mixed with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (potassium salt) (0.2 mg/minute/kg) was infused into the microcirculation of ischemic cremaster by means of intraarterial infusion. Ischemia-reperfusion–induced microcirculatory alterations were measured after 2 hours of reperfusion. Microvasculature of the cremaster muscle including the vascular pedicle was harvested to determine the mitochondrial dysfunction. The blood concentration of methemoglobin was also measured to determine the toxicity of nitrite. Results: The authors found that nitrite significantly attenuated ischemia-reperfusion–induced vasoconstriction, arteriole stagnation, and capillary no-reflow in the early phase of reperfusion and the depolarization of mitochondrial membrane potential and cytochrome c release in the late phase of reperfusion. Nitrite-induced protection was significantly blocked by a nitric oxide scavenger (potassium salt). The methemoglobin results showed that the doses of nitrite we used in the present study were safe. Conclusion: The supplementation of a low dose of nitrite, directly into the microcirculation of ischemic muscle through local intraarterial infusion, significantly attenuated ischemia-reperfusion–induced microcirculatory alterations in vivo and mitochondrial dysfunction in vitro in the microvasculature of skeletal muscle.

The Effect of Lipoaspirates Cryopreservation on Adipose-Derived Stem Cells

BACKGROUND Autologous fat grafting has gained popularity, particularly with the discovery of adipose-derived stem cells (ADSC). The possibility of freezing lipoaspirates (LA) for later use has intriguing clinical potential. However, the effect of LA cryopreservation on ADSC is unclear. OBJECTIVES The authors explore the effect of LA cryopreservation on ADSC viability. METHODS Human LA (n = 8) were harvested using a standard technique. Lipoaspirate samples were either processed immediately as fresh LA (A) or stored at -20°C and then at -80°C for 30 days with (B) or without (C) freezing medium. Stromal vascular fraction (SVF) was separated from adipocytes and either cultured to obtain purified ADSC or processed for the isolation of 3 distinct ADSC subpopulations (CD90(+)/CD45(-), CD105(+)/CD45(-), and CD34(+)/CD31(-)). Apoptosis and necrosis were determined by an annexin V/propidium iodide assay and quantified by flow cytometry. The capability of ADSC for long-term proliferation and differentiation was also examined. RESULTS There were no significant differences in the apoptosis and necrosis of adipocytes, SVF, or ADSC between groups A and B. However, cell viability in SVF and ADSC was significantly compromised in group C as compared with group B (P < .01) due to higher ADSC apoptosis but not necrosis. The viable ADSC isolated from fresh or frozen LA were cultured for more than 20 passages and demonstrated similar patterns and speed of proliferation with strong capability to differentiate, evidenced by cell doubling time and positive staining with Oil Red O (Sigma-Aldrich, St Louis, Missouri) and alkaline phosphatase. CONCLUSIONS Lipoaspirates cryopreservation had a significant impact on ADSC apoptosis but not on ADSC necrosis, proliferation, or differentiations. Freezing medium provides significant protection against ADSC apoptosis.

Attenuating tumor necrosis factor α does not ameliorate other cytokine and peroxidase products during sepsis

BACKGROUND Recent trials utilizing single anticytokine agents have shown no consistent survival benefit in improving the outcome of sepsis. Since an entire cascade of mediators contributes to the underlying pathophysiology, it is not surprising that monotherapy has proven unsuccessful. The purpose of this study was to measure the effects of attenuating tumor necrosis factor (TNF)alpha early in sepsis. METHODS Three groups of Sprague-Dawley rats were studied. All animals were infused with live Escherichia coli, with group I and group II rats additionally receiving a matrix metalloproteinase inhibitor. Serum levels of TNFalpha, interleukin (IL)-6, malondialdehyde (MDA), and lipid hydroperoxide (LOOH) were compared. RESULTS TNFalpha showed a significant decrease, yet IL-6, MDA, and LOOH (markers of sepsis) levels remained abnormally elevated. CONCLUSION Despite significantly attenuating TNFalpha, the septic response continued. This supports the concept that in sepsis, monotherapy directed at attenuating a single cytokine cannot overcome the tissue-damaging effects of an entire cascade of mediators.

Elimination of reperfusion-induced microcirculatory alterations in vivo by adipose-derived stem cell supernatant without adipose-derived stem cells.

Background: In the present study, the authors hypothesized that adipose-derived stem cells in cell culture may secrete multiple cytokines in the supernatant, which might have a significant impact in vivo on the reperfusion-induced microcirculatory alterations and endothelial dysfunction. Methods: Fat tissue was surgically harvested from rat flanks and processed for adipose-derived stem cell isolation; cells (1 × 106) were subcultured for 3, 6, 9, and 12 days without passage. The postcultivated medium was harvested with medium change every 3 days. After centrifugation, the supernatant was collected and stored at −20°C. Supernatant collected on day 9 was analyzed for eight oxidative stress cytokines by an enzyme-linked immunosorbent assay strip. The effect of the supernatant on the reperfusion-induced microcirculatory alterations was examined in the vascular pedicle of isolated rat cremaster muscles subjected to 4 hours of ischemia followed by 2 hours of reperfusion. Results: Enzyme-linked immunosorbent assay results demonstrated that adipose-derived stem cells produced several highly expressed cytokines in the supernatant. The average concentration of interleukin-6, in particular, was 5-fold higher compared with control. The reperfusion-induced vasospasm, arteriole stagnation, and the capillary no-reflow that often appear in the early phase of reperfusion were eliminated by adipose-derived stem cell supernatant. Conclusions: Adipose-derived stem cells in cell culture display cytokine secretory properties that enable the cells to act through paracrine signaling. The supernatant even without cells could be used as a paracrine agent to interfere with the reperfusion–induced microcirculatory alterations and endothelial dysfunction.

The impact of short-term refrigeration of human lipoaspirate on adipose-derived stem cells and adipocytes

Both liposuction and fat grafting are popular procedures in aesthetic surgery. Current clinical practice is that fat grafting has to be conducted immediately after liposuction in the concern that cell death in lipoaspirate could increase as the storage time is prolonged. However, there is no scientific data in the literature either to support or oppose that concern. It has been a strong desire of both surgeons and patients to be able to preserve lipoaspirate for potential future applications. In our previous studies, the viability of adipose-derived stem cells (ASCs) and adipocytes in both fresh and deep-frozen lipoaspirates has been examined and cell apoptosis and necrosis have been quantified by Annexin-V/PI assay and analyzed by flow cytometer. The purpose for the present study was to determine the impact of short-term refrigeration of lipoaspirate on ASCs and adipocytes. Two quantitated functional assays were selected to determine cell viability based on the concept that viable cell does not necessarily mean functional, but the functional cell must be viable. Human lipoaspirates were harvested using standard technique. Each lipoaspiratewas divided into 5 portions with 5 ml of each. The fresh portion was processed immediately and served as control. Other portions were stored in the refrigerator (2e8 C) and processed at 24 h, 48 h, 72 h or 96 h later respectively. The viability of ASCs was determined by the number of ASC after 24 h culture of stromal vascular fraction (SVF). The viability of adipocytes was assessed by glycerol-3-phosphate dehydrogenase (G3PDH) activitywhich is widely used to evaluate the biosynthesis of fat in adipocytes. The sterility of lipoaspirate was assessed by bacterial culture using LB Agar powder and inoculating loop.

Matrix metalloproteinase inhibition protects hepatic integrity in hemorrhagic shock.

Hemorrhagic shock increases cytokines, such as tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), and compromises hepatic function and integrity. The production of TNF-α involves a cascade reaction regulated by the enzyme TNF-a convertase. The purpose of this study was to examine the effects of matrix metalloproteinase inhibitor (MMPI) (British Biotech 1101) in vivo on hepatic integrity in a rat model of hemorrhagic shock. Sprague-Dawley rats (n = 26) were divided as follows: hemorrhagic shock (group 1) and hemorrhagic shock plus MMPI (group 2). TNF-a, IL-6, and hepatic membrane potentials (Em) were obtained. The administration of MMPI significantly decreased TNF-a levels (P <0.001) and stabilized the membrane potential at —30 mV as compared to the depolarized membrane potential at -20 mV for hemorrhagic shock without MMPI. IL-6 levels were not affected by the MMPI. This study demonstrates that MMPI decreases TNF-α levels and protects hepatic integrity in hemorrhagic shock, as evidenced by the stabilization of the membrane potential, independent of the mean arterial pressure. The hepatic protection is closely related to the decrease in TNF-a levels seen in the portal circulation.

Tumescent Liposuction without Lidocaine

Background: Our previous study demonstrated that lidocaine has a negative impact on adipose-derived stem cell (ASC) survival. Currently for large-volume liposuction, patients often undergo general anesthesia; therefore, lidocaine subcutaneous anesthesia is nonessential. We hypothesized that removing lidocaine from tumescent might improve stromal vascular fraction (SVF) and ASC survival from the standard tumescent with lidocaine. Ropivacaine is also a commonly used local anesthetic. The effect of ropivacaine on ASC survival was examined. Methods: Adults who underwent liposuction on bilateral body areas were included (n = 10). Under general anesthesia, liposuction on 1 area was conducted under standard tumescent with lidocaine. On the contralateral side, liposuction was conducted under the modified tumescent without lidocaine. Five milliliters of lipoaspirate were processed for the isolation of SVF. The adherent ASCs were counted after 24 hours of SVF culture. Apoptosis and necrosis of SVF cells were examined by Annexin/propidium iodide staining and analyzed by flow cytometry. Results: Average percentage of live SVF cells was 68.0% ± 4.0% (28.5% ± 3.8% of apoptosis and 3.4% ± 1.0% of necrosis) in lidocaine group compared with 86.7% ± 3.7% (11.5% ± 3.1% of apoptosis and 1.8% ± 0.7% of necrosis) in no-lidocaine group (P = 0.002). Average number of viable ASC was also significantly lower (367,000 ± 107) in lidocaine group compared with that (500,000 ± 152) in no-lidocaine group (P = 0.04). No significant difference was found between lidocaine and ropivacaine on ASC cytotoxicity. Conclusions: Removing lidocaine from tumescent significantly reduced SVF and ASC apoptosis in the lipoaspirate. We recommend tumescent liposuction without lidocaine, particularly if patient’s lipoaspirate will be used for fat grafting.

Optimal Fluid Resuscitation: Timing and Composition of Intravenous Fluids

BACKGROUND Recent data suggest that the timing of fluid resuscitation and the type of fluid used to treat hemorrhagic shock contribute to the inflammatory response as well as cell death. METHODS Rats were bled of 40% of their total blood volume and then resuscitated in either early or delayed fashion. Treatment was assigned randomly and consisted of lactated Ringers solution, normal saline, bicarbonate Ringers solution, hypertonic saline, or no resuscitation. The first four groups were subdivided into early and late resuscitation. After a 5-h observation period, lung and liver samples were evaluated for apoptosis, and blood was collected for measurements of the cytokines interleukin (IL)-6, IL-10, and IL-1beta. RESULTS The rats that were not resuscitated had significantly more apoptosis in liver tissue. In the lung, bicarbonate Ringers solution, when given early, was associated with significantly less apoptosis. Non-resuscitated rats had significantly higher IL-6 concentrations than all other groups. Animals receiving hypertonic saline early had significantly higher IL-6 concentrations than those given any other fluid. The concentration of IL-1beta was significantly higher in the non-resuscitated rats than in those receiving bicarbonate Ringers, lactated Ringers, or normal saline for early resuscitation. Interleukin-10 was elevated significantly in non-resuscitated rats. CONCLUSIONS Cellular destruction and a pro-inflammatory response follow hemorrhagic shock. Early resuscitation with isotonic crystalloid fluids decreases these responses.