Louis C. Argenta’s research while affiliated with American Society of Ophthalmic Plastic and Reconstructive Surgery and other places

Our previous studies on the treatment of spinal cord injuries with Mechanical Tissue Resuscitation (MTR) in rats have demonstrated that it can significantly improve the locomotor recovery and BBB scores. MTR treatment also reduced fluid accumulations by T2-imaging and improved the mean neural fiber number and fiber length in injured sites by fiber tractography. Myelin volume was also significantly preserved by MTR treatment. For further clinical application, a large animal model is necessary to assess this treatment. This study examined the effects of application of MTR on traumatic spinal cord injury in a swine model. Traumatic spinal cord contusion injuries (SCI) in swine were created by controlled pneumatic impact device. Negative pressure at -75 mm Hg was continuously applied to the injured site through open cell silicone manifold for 7 days. In vivo MR imaging for T2 and GRE analysis employed a 3T machine, while a 7T machine was employed for diffusion tensor imaging (DTI) and fiber tractography. Histological HE and Luxol fast blue staining were examined. MTR significantly reduced the mean injured volumes over 46% by T2-imaging in the injured sites from 477.34±146.31 mm3 in non-treated group to 255.99±70.28 mm3 in MTR treated group (P<0.01). It also reduced fluid accumulations by relative T2 signal density in the epicenter of the SCI from 1.62±0.27 in non-treated group to 1.22±0.10 in the MTR treated group (P<0.05). The mean injured tissue volume measured by H&E staining was 303.71±78.21 mm3 in the non-treated group and decreased significantly to 162.16±33.0 mm3 in the MTR treated group (P<0.01). The myelin fiber bundles stained by Luxol blue were preserved much more in the MTR treated group (90±29.71 mm3) than in the non-treated group (33.68±24.99 mm3, P<0.01). The fractional anisotropy (FA) values processed by DTI analysis are increased from 0.203±0.027 in the untreated group to 0.238±0.029 in MTR treatment group (P<0.05). Fiber tractography showings the mean fiber numbers across the impacted area were increased over 112% from 327.0±99.74 in the non-treated group to 694.83±297.86 in the MTR treated group (P<0.05). These results indicates local application of MTR for seven days to spinal cord injury in a swine model decreased tissue injury, reduced tissue edema and preserved more myelin fibers as well as nerve fibers in the injured spinal cord. Keywords: Mechanical tissue resuscitation, Negative pressure treatment, Spinal cord injury, Diffusion tensor imaging, Nerve fiber tractography.

OBJECTIVE The previous laboratory and clinical experience of the authors had demonstrated that application of controlled subatmospheric pressure directly to injured soft tissue can result in increased survival of compromised tissues. Mechanical tissue resuscitation (MTR) is a new concept evolving from these discoveries. The authors’ recent studies have demonstrated that traumatic brain injury tissue can also be salvaged. The aim of this study was to examine the effects of MTR application to injuries from intracerebral hemorrhages (ICHs) in a swine model. METHODS The ICHs in swine were simulated by infusion of autologous artery blood into the right frontal lobe. A specially designed silicone manifold device was introduced directly into the hematoma. Continuous negative pressure at −50 mm Hg was applied through this device. T2- and T2*-weighted MRI, histological H&E staining, and immunostaining were examined. RESULTS After 1 week of treatment, MTR significantly decreased gross hematoma volume by more than 60%, from 472.62 ± 230.19 mm ³ in the nontreated group to 171.25 ± 75.38 mm ³ in the MTR-treated group (p < 0.05). Total hypointense volumes measured on T2*-weighted MR images decreased from 791.99 ± 360.47 mm ³ in the nontreated group to 371.16 ± 105.75 mm ³ in the MTR-treated group (p < 0.05). The hyperintense area on the T2-weighted MR image decreased significantly from 2656.23 ± 426.26 mm ³ in the nontreated group to 1816.66 ± 525.26 mm ³ in the MTR-treated group (p < 0.05). When ICHs were treated with MTR for 2 weeks, the gross hematomas were reduced by 94%, from 112.23 ± 66.21 mm ³ in the nontreated group to 6.12 ± 10.99 mm ³ in the MTR-treated group (p = 0.003). MTR significantly decreased the total necrotic tissue volume in H&E staining from 120.42 ± 48.35 mm ³ in the nontreated group to 60.94 ± 38.99 mm ³ in the MTR-treated group (p < 0.05). The total hypointense volumes on T2*-weighted MR images were significantly reduced, from 385.54 ± 93.85 mm ³ in the nontreated group to 220.54 ± 104.28 mm ³ in the MTR-treated group (p < 0.05), while their mean T2 hyperintense volume decreased significantly from 2192.83 ± 728.27 mm ³ in the nontreated group to 1366.97 ± 463.36 mm ³ in the MTR-treated group (p < 0.05). Histology revealed that the capillary diameter in the reactive tissue rim adjacent to the hematoma increased in both the 1- and 2-week MTR-treated groups. Both von Willebrand factor and CD31 signals were detectable in endothelial cells within the hematoma cavity of both MTR-treated groups. CONCLUSIONS This study demonstrates that local continuous application of controlled subatmospheric pressure to an ICH can safely remove more than half of a clot in 1 week and more than 90% in 2 weeks.

Dehydration and brain interstitial fluid alterations associate to cognitive dysfunction. We now explore whether changes in matrix hydration are a possible common signal for modulation of water-transfer rates and neuron function.

Aging in humans and animals is associated with gradual and variable changes in some cognitive functions, but what causes them and explains individual variations remains unclear. Hydration decreases with aging but whether dehydration contributes to cognitive dysfunction is not known. The brain hydration of aging mice was determined by colloidosmotic-pressure titration. Dehydration increased with age from ∼76 mmHg at 6 weeks to ∼105 mmHg at 40 weeks, or a progressive ∼10 percent loss of brain water but seemed to level off afterward. When we adjusted dehydration in hippocampal slices of <8-week-old mice to the levels seen in mice 40 weeks and older, their basal synaptic responses were amplified at all stimulus voltages tested, but induction of late-phase long-term potentiation was impaired. Our results document progressive brain dehydration with age in inbred mice to levels at which in vitro synaptic plasticity appears dysregulated. They also suggest that dehydration contributes to some of the changes in synaptic plasticity observed with aging, possibly due to adjustments in neuronal excitation mechanisms.

Dehydration impairs cognitive function. To explore the possible mechanisms, we dehydrated mouse brain tissue and measured LTP, the best understood cellular process of synaptic plasticity that underlies learning and memory in vertebrates. We determined physiologic hydration by titrating water activity in mouse brain explants. Specifically, we immersed the 60–90‐mg explants in baths of artificial cerebrospinal fluid (ACSF) equilibrated at room temperature in which inert, non‐penetrating polymer polyethylene glycol 8000 was dissolved to attain colloidosmotic pressures of 0 mmHg (in control ACSF), 54 mmHg, 101 mmHg, and 196 mmHg. We then calculated the parameters reflecting brain hydration, including hydration potential and hydraulic conductance, from initial flow rates determined gravimetrically. To explore the effect of dehydration on synaptic plasticity, hippocampus slices were equilibrated for 30 min at the above pressures prior to recording field excitatory postsynaptic potentials (fEPSP). Parameters for basal synaptic transmission were derived from input‐output tracings following incremental stimuli. In a separate set of slices, L‐LTP was induced by spacing four 100‐Hz trains 5‐min apart and recording the results for 3 h. Water activity titrations were conducted at room temperature and LTP at 32 °C. All experiments were reproduced in at least 6 mice; results are presented as means and standard errors. Fluid transferred either to or from the brain explants, depending on the bath pressure. Initial fluid transfer rates—3.486 ± 0.1919, 0.08950 ± 0.3907, −1.820 ± 1.258 and −4.248 ± 2.338 μl/min/g— increased linearly ( r ² > 0.9, by linear regression analyses ) with nominal bath pressures of 0, 54, 101, and 196 mmHg, respectively. The hydraulic conductance , calculated from the slope of the fitted regression lines, was 0.051 ± 0.0019 μl/min/g/mmHg, and the hydration potential , calculated from the pressure at initial rate = 0, was 56.44 ± 4.96 mmHg. In slices equilibrated at near this physiologic hydration potential, basal synaptic transmission resembled that of the slices in control ACSF. However, dehydrating the slices by increasing the colloid osmotic pressure increased basal synaptic transmission, indicating neuronal hyper‐excitability. Similarly, L‐LTP induced at the 54 mmHg level did not differ from that in controls, but progressive dehydration impaired its induction. Induction was completely abolished at a nominal pressure of 196 mmHg. The surprisingly high hydration potential measured in the explanted brain tissue indicates lower water activity in the brain interstitial matrix than in cerebrospinal fluid (CSF) and reflects competition for water in the crowded brain microenvironment. As a notable consequence, neuronal excitability is highly sensitive to changes in water activity. Our findings predict that local dehydration affects cognitive function and impairs synaptic plasticity by preventing L‐LTP induction.

The interstitial myocardial matrix is a complex and dynamic structure that adapts to local fluctuations in pressure and actively contributes to the heart’s fluid exchange and hydration. However, classical physiologic models tend to treat it as a passive conduit for water and solute, perhaps because local interstitial regulatory mechanisms are not easily accessible to experiment in vivo. Here, we examined the interstitial contribution to the fluid-driving pressure ex vivo. Interstitial hydration potentials were determined from influx/efflux rates measured in explants from healthy and ischemia-reperfusion-injured pigs during colloid osmotic pressure titrations. Adaptive responses were further explored by isolating myocardial fibroblasts and measuring their contractile responses to water activity changes in vitro. Results show hydration potentials between 5 and 60 mmHg in healthy myocardia and shifts in excess of 200 mmHg in edematous myocardia after ischemia-reperfusion injury. Further, rates of fluid transfer were temperature-dependent, and in collagen gel contraction assays, myocardial fibroblasts tended to preserve the micro-environment’s hydration volume by slowing fluid efflux rates at pressures above 40 mmHg. Our studies quantify components of the fluid-driving forces in the heart interstitium that the classical Starling’s equation does not explicitly consider. Measured hydration potentials in healthy myocardia and shifts with edema are larger than predicted from the known values of hydrostatic and colloid osmotic interstitial fluid pressures. Together with fibroblast responses in vitro, they are consistent with regulatory mechanisms that add local biological controls to classic fluid-balance models.

Neuronal function depends on exquisite local regulation of a great variety of molecules and their interactions in the interstitial spaces. The timing and magnitude of interactions in the brain must depend on the hydraulic properties governing water and solute transfer across its matrix, but quantitative information on these properties has proved notoriously difficult to obtain, and uncertainties remain. Here, we adapt osmotic stress techniques to explore pressure-volume relationships and fluid-transfer parameters in brain tissue. Within 15-30 minutes of euthanasia, sets of brain tissue samples (12/brain, ∼1g each) were obtained from pigs (n =11) and immersed in baths adjusted to colloidosmotic pressures of 3-219 mmHg with polyethyleneglycol Mw 8000. The samples’ water influx/efflux was measured gravimetrically as a function of time, normalized by weight. Initial flow rates were calculated from the first derivative, at time = 0, of second-degree polynomials fitted to the progress curves. Rates were linearly proportional to the bath's pressure (r2 > 0.9). The hydraulic conductance calculated from the slope of fitted lines was 0.029 ± 0.008µl/min/g/mmHg, and the hydration potential, calculated from the pressure at initial rate = 0, was 64 ± 27 mmHg (means ± SD, n = 11).The hydraulic conductance, but not the hydration potential, decreased significantly when the bath temperature was reduced from 37 to 4°C. Compared to heart and skin, brain conductance value was higher. Further, the changes in hydraulic parameters with temperature were distinct for each organ. These results illustrate a simple, ex vivo, quantitative approach to probe specific water-transfer parameters and their changes in the brain. They indicate organ-dependent differences in flow regulation and the hydraulic properties of extracellular matrices.