Sex and region-specific effects of high fat diet on PNNs in obesity susceptible rats.

Perineuronal nets (PNNs) are specialized extracellular matrix structures that primarily surround fast-spiking parvalbumin (PV)-containing interneurons within the PFC. They regulate PV neuron function and plasticity to maintain cortical excitatory/inhibitory balance. For example, reductions in PNN intensity are associated with reduced local inhibition and enhanced pyramidal neuron firing. We previously found that exposure to dietary high fat reduced PNN intensity within the PFC of male Sprague-Dawley (SD) rats. However, how high fat affects PNNs in the PFC of females or in obesity-vulnerable vs. -resistant models is unknown. Therefore, we gave male and female SD, selectively bred obesity-prone (OP), and obesity-resistant rats (OR) free access to standard lab chow or 60% high fat for 21 days. We then measured the number of PNN positive cells and PNN intensity (determined by Wisteria floribunda agglutinin [WFA] staining) as well as the number of PV positive neurons using immunohistochemistry. We found sex and region-specific effects of dietary high fat on PNN intensity, in the absence of robust changes in cell number. Effects were comparable in SD and OP but differed in OR rats. Specifically, high fat reduced PNN intensities in male SD and OP rats but increased PNN intensities in female SD and OP rats. In contrast, effects in ORs were opposite, with males showing increases in PNN intensity and females showing a reduction in intensity. Finally, these effects were also region specific, with diet-induced reductions in PNN intensity found in the prelimbic PFC (PL-PFC) and ventral medial orbital frontal cortex (vmOFC) of SD and OP males in the absence of changes in the infralimbic PFC (IL-PFC), and increases in PNN intensity in the IL-PFC of SD and OP females in the absence of changes in other regions. These results are discussed in light of roles PNNs may play in influencing PFC neuronal activity and the differential role of these sub-regions in food-seeking and motivation.

Consumption of a High-Fat Diet Alters Perineuronal Nets in the Prefrontal Cortex.

A key factor in the development of obesity is the overconsumption of fatty foods, which, in addition to facilitating weight gain, alters neuronal structures within brain reward circuitry. Our previous work demonstrates that sustained consumption of a high-fat diet (HFD) attenuates spine density in the prefrontal cortex (PFC). Whether HFD promotes structural adaptation among inhibitory cells of the PFC is presently unknown. One structure of interest is the perineuronal net (PNN), a specialized extracellular matrix surrounding, primarily, parvalbumin-containing GABAergic interneurons. PNNs contribute to synaptic stabilization, protect against oxidative stress, regulate the ionic microenvironment within cells, and modulate regional excitatory output. To examine diet-induced changes in PNNs, we maintained rats on one of three dietary conditions for 21 days: ad libitum chow, ad libitum 60% high fat (HF-AL), or limited-access calorically matched high fat (HF-CM), which produced no significant change in weight gain or adiposity with respect to chow controls. The PNN "number" and intensity were then quantified in the prelimbic (PL-PFC), infralimbic (IL-PFC), and ventral orbitofrontal cortex (OFC) using Wisteria floribunda agglutinin (WFA). Our results demonstrated that fat exposure, independent of weight gain, induced a robust decrease in the PNN intensity in the PL-PFC and OFC and a decrease in the PNN number in the OFC.

Photoperiodic suppression of drug reinstatement.

The rewarding influence of drugs of abuse varies with time of day and appears to involve interactions between the circadian and the mesocorticolimbic dopamine systems. The circadian system is also intimately involved in measuring daylength. Thus, the present study examined the impact of changing daylength (photoperiod) on cocaine-seeking behaviors. Male Sprague-Dawley rats were trained and tested on a 12L:12D light:dark schedule for cocaine-induced reinstatement of conditioned place preference (CPP) at three times of day (Zeitgeber time (ZT): 4, 12, and 20) to determine a preference score. Rats were then shifted to either shorter (6L:18D) or longer (18L:6D) photoperiods and then to constant conditions, re-tested for cocaine-induced reinstatement under each different condition, and then returned to their original photoperiod (12L:12D) and tested once more. Rats exhibited a circadian profile of preference score in constant darkness with a peak at 12 h after lights-off. At both ZT4 and ZT20, but not at ZT12, shorter photoperiods profoundly suppressed cocaine reinstatement, which did not recover even after switching back to 12L:12D. In contrast, longer photoperiods did not alter reinstatement. Separate studies showed that the suppression of cocaine reinstatement was not due to repeated testing. In an additional experiment, we examined the photoperiodic regulation of tyrosine hydroxylase (TH) and dopamine transporter (DAT) proteins in drug-naive rats. These results revealed photoperiodic modulation of proteins in the prefrontal cortex and dorsal striatum, but not in the nucleus accumbens or ventral tegmental area. Together, these findings add further support to the circadian genesis of cocaine-seeking behaviors and demonstrate that drug-induced reinstatement is modulated by photoperiod. Furthermore, the results suggest that photoperiod partly contributes to the seasonal expression of certain drug-related behaviors in humans living at different latitudes and thus our findings may have implications for novel targeting of circadian rhythms in the treatment of addiction.

Activation of dopamine D1 receptors in the medial prefrontal cortex produces bidirectional effects on cocaine-induced locomotor activity in rats: effects of repeated stress.

We examined the effects of repeated stress and D1 receptor activation in the medial prefrontal cortex (mPFC) on acute-cocaine-induced locomotor activity in rats. Male rats were given 7 days of either handling (Controls) or a variety of stressors. After 8-17 days' withdrawal, rats received an intra-mPFC microinjection of the full D1 agonist, SKF 81297: 0, 0.03, 0.1 or 0.3 microg/side followed by an i.p. saline or cocaine injection (15 mg/kg, i.p.). The target sites were either the dorsal or ventral mPFC. We also divided rats into either high or low responders based on their locomotor response to an acute cocaine injection. In the dorsal PFC, low responder Control and Stress groups demonstrated an augmentation of cocaine-induced increases in activity after SKF 81297, compared with vehicle, microinjection. In contrast, high responder rats demonstrated a suppression of cocaine-induced increases in activity after intra-mPFC SFK 81297 infusion, with an apparent 10 times higher sensitivity in the Stress group. In the ventral PFC, low responder Controls showed no changes after SKF 81297 infusion, while the Stress group showed an increase in cocaine-induced activity in response to SKF 81297. In high responders given SFK 81297 into the ventral mPFC, cocaine-induced activity was suppressed in Controls, while stress pretreatment rendered animals resistant to SKF 81297 effects. These results indicate that D1 receptor activation effects in the mPFC are bidirectional depending on whether rats have a high or low locomotor response to cocaine. Further, daily stress alters the sensitivity of the mPFC to SKF 81297, which is dependent on whether the dorsal or ventral mPFC is targeted.

Manipulation of dopamine d1-like receptor activation in the rat medial prefrontal cortex alters stress- and cocaine-induced reinstatement of conditioned place preference behavior.

These studies examined the ability of the dopamine D1-like agonist SKF 81297 and D1-like antagonist SCH 23390 in the medial prefrontal cortex to alter the reinstatement of cocaine-induced conditioned place preference behavior. Male Sprague-Dawley rats were fitted with bilateral cannulae over the medial prefrontal cortex and subsequently trained in a conditioned place preference task. Animals were trained in this task using four pairings of cocaine (12 mg/kg, i.p.). Conditioned place preference was demonstrated in all animals, and this behavior was then extinguished over a 5-10-day period before testing for reinstatement. Just prior to reinstatement by immobilization stress or a cocaine priming injection (5 mg/kg, i.p.), a microinjection of the D1-like receptor antagonist SCH 23390 (0.01, 0.1 or 1.0 microg/side), or the D1-like receptor agonist SKF 81297 (0.1, 0.3 or 1.0 microg/side) was given into the medial prefrontal cortex. SCH 23390 blocked both stress- and cocaine-induced reinstatement of conditioned place preference after the two higher doses were administered into the medial prefrontal cortex. The highest dose of SKF 81297 (1.0 microg/side) prevented immobilization stress- but not cocaine-induced reinstatement. The highest dose of these drugs given in the absence of stress or cocaine did not produce reinstatement. The results indicate that immobilization stress given within the place-preference chamber is capable of producing reinstatement of cocaine-seeking behavior. The microinjection studies suggest that D1-like receptor antagonism within the prefrontal cortex is sufficient to block reinstatement by stress and cocaine. Furthermore, the results from D1-like receptor activation in the medial prefrontal cortex point to utilization of different neural pathways for stress- and cocaine-induced reinstatement.

Regulation of medial prefrontal cortex dopamine by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors.

In the medial prefrontal cortex, repeated cocaine produces tolerance of the extracellular dopamine response to subsequent cocaine injection. These studies characterized the influence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors on the medial prefrontal cortex dopamine response to acute cocaine, amphetamine and potassium chloride as a first step to assess whether these receptor subtypes may be candidates for mediating dopamine tolerance after repeated cocaine. Local infusion of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) produced an approximate 40% increase in dopamine levels in the medial prefrontal cortex, while a 30 microM dose did not alter basal levels infused over a 3-h period. Thus, 30 microM CNQX was chosen for the remaining experiments, and was infused for 1 h prior to and during all in vivo treatments. Local medial prefrontal cortex infusion of the 30 microM dose blocked the small increase in dopamine levels elicited by systemic saline injection (maximum of 26%), as well as the much larger increase in response to acute cocaine injection (maximum of 340%). Local infusion of D-amphetamine (3 and 30 microM) through the probe increased dopamine to 300 and 600% of basal levels, respectively. Co-infusion of CNQX partially blocked the response for the first 40 min, but dopamine levels recovered by 60 min later. Local infusion of 100 mM potassium chloride elicited a 600% increase in dopamine levels, which was attenuated approximately 50% by CNQX co-infusion. Potassium-stimulated release of dopamine was also measured in vitro in medial prefrontal cortical and striatal tissue. By 30 s after potassium addition, dopamine levels increased to 800% above baseline in the medial prefrontal cortex, and this increase was blocked by the presence of 30 microM CNQX. In contrast, potassium-stimulated dopamine release in striatal tissue was approximately 250% above basal levels, with no effect of CNQX on dopamine release. Locomotor behavior collected during dialysis experiments demonstrated that increased activity induced by local infusion of potassium chloride was severely attenuated by co-infusion of 30 microM CNQX, while no effects of this drug were found for cocaine-elicited behavior. These results suggest a potent influence of glutamate via alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors on extracellular dopamine in the medial prefrontal cortex, and these receptors may regulate dopamine release through a presynaptic mechanism. The findings may help elucidate the role of medial prefrontal cortex dopamine-glutamate interactions in drug abuse and stress- and drug-precipitated psychosis.

Conditioned fear stimuli reinstate cocaine-induced conditioned place preference.

These studies examined the ability of a conditioned stimulus previously paired with footshock to reinstate cocaine-induced conditioned place preference. Male rats were given either odor or tone in a paired (PRD group) or explicitly unpaired (random, RND group) manner with footshock. All rats were subsequently trained in a cocaine conditioned place preference (CPP) task. Cocaine CPP was demonstrated in all groups. After CPP extinction, presentation of the conditioned fear stimulus produced a greater degree of reinstatement in PRD rats compared to the RND group. This was true whether the conditioned stimulus was odor or tone, but when odor was used as the conditioned stimulus, the RND group also partially reinstated cocaine CPP. In rats trained with tone as the conditioned stimulus, presentation of the tone during the test for reinstatement produced robust reinstatement of cocaine CPP only in the PRD, but not RND, group. In contrast, a subsequent priming injection with cocaine reinstated cocaine CPP equally in both RND and PRD groups. These studies indicate for the first time that conditioned fear stimuli induce reinstatement of cocaine CPP, and suggest that stimuli associated with prior stress may produce relapse in humans.

Laser-tissue soldering with biodegradable polymer films in vitro: film surface morphology and hydration effects.

BACKGROUND AND OBJECTIVE: Previous research introduced the concept of using biodegradable polymer film reinforcement of a liquid albumin solder for improvement of the tensile strength of repaired incisions in vitro. In this study, the effect of creating small pores in the PLGA films on the weld breaking strength is studied. Additionally, the effect of hydration on the strength of the reinforced welds is investigated. STUDY DESIGN/MATERIALS AND METHODS: A 50%(w/v) bovine serum albumin solder with 0.5 mg/mL Indocyanine Green dye was used to repair an incision in bovine aorta. The solder was coagulated with an 806-nm CW diode laser. A poly(DL-lactic-co-glycolic acid) (PLGA) film was used to reinforce the solder (the controls had solder but no reinforcement). Breaking strengths were measured acutely and after hydration in saline for 1 and 2 days. The data were analyzed by ANOVA (P < 0.05) and multiple comparisons of means were performed using the Newman-Keuls test. RESULTS: The creation of pores in the PLGA films qualitatively improved the film flexibility without having an apparent adverse effect on the breaking strength, while the actual technique of applying the film and solder had more of an effect. The acute maximum average breaking strengths of some of the film reinforced specimens (114.7 g-134.4 g) were significantly higher (P < 0.05) than the acute maximum average breaking strength of the unreinforced control specimens (68.3 g). Film reinforced specimens were shown to have a statistically significantly higher breaking strength than unreinforced controls after 1- and 2-day hydration. CONCLUSIONS: Reinforcement of liquid albumin solders in laser-assisted incision repair appears to have advantages over conventional methods that do not reinforce the cohesive strength of the solder in terms of acute breaking strength and after immersion in moist environments for short periods of time. Using a film with the solder applied to one surface only may be advantageous over other techniques.

New range of light-activated surgical adhesives for tissue repair.

A new range of light-activated surgical adhesives has been developed for laser-assisted tissue repair. The surgical adhesive is composed of a polymer scaffold doped with the traditional protein solder mix of serum albumin and a chromophoric dye. Problems associated with inflexibility in conforming to various tissue geometries, instability in a hydrated environment due to non-uniform tissue adhesive denaturation, and solubility in physiological fluids prior to denaturation are overcome using the adhesives. The new adhesives offer a viable alternative to conventional fasteners, including sutures, staples and clips, currently used for tissue repair. In addition, it could be possible to use patches prepared from the adhesive in the field as a simple and effective method to stop bleeding and repair tissue quickly in an emergency situation. First, studies were conducted to improve the flexibility of traditional protein solders to enable them to be tailored to a wide range of clinically relevant geometry's including tubes, crescents and tape. Second, the creation of a chromophore concentration gradient across the thickness of the adhesive was investigated as a means to allow control of the heat source gradient through the adhesive. Increased deposition of the laser energy near the vital solder/tissue interface was thus achieved. Finally, predenaturation of the adhesive was investigated as a means for enhancing its stability in a hydrated environment thus improving the handling characteristics of the adhesive for clinical application. The application of the new surgical adhesives to augment laser tissue repairs is shown to enhance edge co-optation, improve repair strength and to reduce thermal tissue injury. The moldable, absorption controllable and flexible nature of the new adhesives greatly improves the clinical applicability of laser-solder tissue repair.

Dopamine D1 receptor activation in the medial prefrontal cortex prevents the expression of cocaine sensitization.

This study examined whether microinjection of the full D1 agonist, SKF 81297, or the D1 antagonist, SCH 23390, into the medial prefrontal cortex (mPFC) would alter the expression phase of cocaine sensitization. Male Sprague-Dawley rats were administered saline or cocaine (15 mg/kg, i.p.) once per day for seven consecutive days. After 8 to 17 days withdrawal, rats received a bilateral intra-mPFC microinjection of SKF 81297: either 0, 0.03, 0.1, or 0.3 microg/side; SCH 23390: either 0, 0.1, 0.3, or 1.0 microg/side; or a combination of 0.1 microg of SKF 81297 + 0.3 microg of SCH 23390, followed by an i.p. saline or cocaine (15 mg/kg, i.p.) injection. In naïve rats, vertical activity was elevated by the two lower doses of SKF 81297. A similar enhancement of cocaine-induced activity was observed in daily saline rats at the highest dose tested. In contrast, SKF 81297 suppressed the expression of sensitization to cocaine. This blockade of sensitization was prevented by coinfusion of SCH 23390. Infusion of SCH 23390 alone into the mPFC in daily saline and cocaine-pretreated rats demonstrated a suppression of cocaine-induced locomotion in daily saline-pretreated rats after the highest dose, but a slight augmentation of activity after the lowest dose in daily cocaine-pretreated rats. These results demonstrate a contribution by mPFC D1 receptors in the expression of cocaine sensitization and further suggest that the effects of D1 receptor activation in the mPFC occur in opposite directions in daily saline versus daily cocaine-pretreated rats.

Exposure to repeated low-level formaldehyde in rats increases basal corticosterone levels and enhances the corticosterone response to subsequent formaldehyde.

Low-level exposure to volatile organic compounds may produce symptoms in humans reporting multiple chemical sensitivity (MCS) through altered hypothalamic-pituitary-adrenal (HPA) axis functioning. We determined whether repeated formaldehyde (Form) exposure would alter corticosterone (CORT) levels in a rat model of MCS. Male Sprague-Dawley rats were given acute chamber exposures to Air or Form (0.7 or 2.4 ppm), and trunk blood was collected 20 or 60 min later. All groups showed increased CORT levels above naïve basal levels at 20 min and a return to baseline by 60 min, with no differences between treatment groups. The second experiment examined the effect of repeated Form exposure (1 h/day x 5 days/week x 2 or 4 weeks) on basal CORT levels and after a final challenge. Basal CORT was increased above naïve values after 2 week exposure to Air or 0.7 ppm Form. By 4 week, CORT levels in the Air group returned to naïve values, but remained elevated in the 0.7 ppm Form group. There were no differences in basal CORT levels among either 2.4 ppm exposed groups. After a final Air or Form challenge, the 2 and 4 week Air and 0.7 ppm Form groups had elevated CORT levels similar to their acute response, while the 2 and 4 week 2.4 ppm Form groups had elevated CORT levels compared to their acute response, indicating enhanced reactivity of the HPA axis to subsequent Form. These findings suggest that altered HPA axis functioning occurs after repeated low-level Form exposure, and may have implications for mechanisms mediating MCS in humans.

Improved vascular tissue fusion using new light-activated surgical adhesive on a canine model.

Newly developed light-activated surgical adhesives have been investigated as a substitute to traditional protein solders for vascular tissue fusion without the need for sutures. Canine femoral arteries (n = 14), femoral veins (n = 14), and carotid arteries (n = 10) were exposed, and a 0.3-0.6 cm longitudinal incision was made in the vessel walls. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of bovine serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805 nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The new adhesives were flexible enough to be wrapped around the vessels while their solid nature avoided the problems associated with "runaway" of the less viscous liquid protein solders widely used by researchers. Assessment parameters included measurement of the ex vivo intraluminal bursting pressure 1-2 h after surgery, as well as histology. The acute intraluminal bursting pressures were significantly higher in the laser-solder group (>300 mmHg) compared to the suture control group (<150 mmHg) where four evenly spaced sutures were used to repair the vessel (n = 4). Histological analysis showed negligible evidence of collateral thermal damage to the underlying tissue in the laser-solder repair group. These initial results indicated that laser-assisted vascular repair using the new adhesives is safe, easy to perform, and contrary to conventional suturing, provides an immediate leak-free closure. In addition, the flexible and moldable nature of the new adhesives should allow them to be tailored to a wide range of tissue geometries, thus greatly improving the clinical applicability of laser-assisted tissue repair.

Dietary cancer risk from conditional cancerogens (tumor promoters) in produce of livestock fed on species of spurge (Euphorbiaceae). V. Skin irriitant and tumor-promoting diterpene ester toxins of the tigliane and ingenane type in the herbs Euphorbia nubica and Euphorbia helioscopia contaminating fodder of livestock.

Irritant diterpene ester toxins were isolated from Euphorbia nubica and E. helioscopia, which are contaminants of the green fodder of livestock in Egypt. Fractionations of methanol extracts of aerial parts of both plants were monitored by the irritation unit on the mouse ear. Plant extracts were subjected to multiplicative distribution methods, yielding irritant hydrophilic fractions that were further purified by column chromatography. Final purification of the materials was achieved by TLC (silica gel) followed by HPLC, or by TLC alone. In this way, from E. nubica, five Euphorbia factors (Nu1-Nu5) were isolated and characterized as short-chain polyfunctional diterpene esters of tigliane-type parent alcohols. The two weak irritants Nul and Nu3 were triesters of 4-deoxy(4alpha)phorbol. Nu2 was shown to be a triester of the stereoisomeric tigliane-type parent alcohol 4-deoxyphorbol. Weak irritant Nu4 probably is a positional isomer of Nu2. Nu5 was characterized as a short-chain triester of 4,20-dideoxy-5xi-hydroxyphorbol. From E. helioscopia, six short- to medium-chain polyfunctional diterpene esters of the ingenane type, generally containing unsaturated acids were obtained, i.e., four irritant esters of ingenol (Euphorbia factors H1, H2, H5, and H6) and two esters of 20-deoxyingenol (non-irritant Euphorbia substance HS4, and irritant Euphorbia factor H8). All irritant Euphorbia factors of the tigliane and ingenane diterpene ester type described in this investigation are considered to be more or less active tumor promoters, i.e., conditional (non-genotoxic) cancerogens. The Euphorbia factors assayed exhibited moderate (H1) to low (H8) relative tumor-promoting potency in comparison to the ingenane prototype DTE tumor promoter 3-TI.

Characterization of extracellular dopamine clearance in the medial prefrontal cortex: role of monoamine uptake and monoamine oxidase inhibition.

In vitro rotating disk electrode (RDE) voltammetry and in vivo microdialysis were used to characterize dopamine clearance in the rat medial prefrontal cortex (mPFC). RDE studies indicate that inhibition by cocaine, specific inhibitors of the dopamine transporter (DAT) and norepinephrine transporter (NET), and low Na(+) produced a 50-70% decrease in the velocity of dopamine clearance. Addition of the monoamine (MAO) inhibitors, l-deprenyl, clorgyline, pargyline, or in vivo nialamide produced 30-50% inhibition. Combined effects of uptake inhibitors with l-deprenyl on dopamine clearance were additive (up to 99% inhibition), suggesting that at least two mechanisms may contribute to dopamine clearance. Dopamine measured extracellularly 5 min after exogenous dopamine addition to incubation mixtures revealed that most conditions of DAT/NET inhibition did not produce elevated dopamine levels above controls. Inhibition of MAO produced elevated dopamine levels only after long-term, but not short-term, incubation in vitro. Short-term incubation of l-deprenyl combined with DAT and NET uptake inhibitors increased dopamine above control levels, consistent with more than one mechanism of dopamine clearance. Local infusion of pargyline (100 or 300 microm) into the mPFC or striatum via microdialysis produced more pronounced and immediate increases in mPFC dopamine levels compared with striatum. Furthermore, dopamine elevation in the mPFC was not accompanied by a decrease in the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, as found in the striatum. These findings may have revealed a unique mechanism of mPFC dopamine clearance and therefore contribute to the understanding of multiple behaviors that involve mPFC dopamine transmission, such as schizophrenia, drug abuse, and working memory function.

Repeated formaldehyde effects in an animal model for multiple chemical sensitivity.

Chemical intolerance is a phenomenon observed in multiple chemical sensitivity (MCS) syndrome, an ill-defined disorder in humans attributed to exposure to volatile organic compounds. Amplification of symptoms in individuals with MCS resembles the phenomenon of psychostimulant- and stress-induced sensitization in rodents. We have recently tested in rats the hypothesis that repeated chemical exposure produces sensitization of central nervous system (CNS) circuitry. A rat model of MCS in our laboratory has employed several endpoints of CNS function after repeated formaldehyde (Form) exposure (1 h/day x 5 days/week x 4 weeks). Repeated Form exposure produced behavioral sensitization to later cocaine injection, suggesting altered dopaminergic sensitivity in mesolimbic pathways. Rats given repeated Form also demonstrated increased fear conditioning to odor paired with footshock, implicating amplification of neural circuitry guiding fear responding to a conditioned odor cue. Recent studies examining the effects of repeated Form on locomotor activity during each daily exposure showed a decrease in rearing activity after 12-15 days of Form exposure compared to air-exposed controls. EEG recordings taken 1 week after withdrawal from daily Form revealed altered sleep architecture. Some of the differences in sleep disappeared after subsequent brief (15 min) challenge with Form the next day. Overall, the findings indicate that repeated low-level chemical exposure produces behavioral changes that may be akin to those observed in individuals with MCS, such as greater sensitivity to chemicals manifest as increased anxiety upon chemical exposure and altered sleep and/or fatigue. Study of the underlying CNS changes will provide a basis for mechanistically based animal models for MCS.

Novel solid protein solder designs for laser-assisted tissue repair.

BACKGROUND AND OBJECTIVES: Previous studies have shown that the application of chromophore-enhanced albumin protein solders to augment laser tissue repairs significantly improves repair strength, enhances edge co-optation, and reduces thermal tissue injury. These investigations are furthered with this in vitro study conducted to assess a new range of specially designed chromophore-enhanced solid protein solders manufactured and tested for application during laser-assisted tissue repair. STUDY DESIGN/MATERIALS AND METHODS: The experimental study was divided into three parts. In the first part of the study, the creation of a chromophore concentration gradient across the thickness of the solid protein solder was investigated as a means to improve control of the heat source gradient through the solder during laser irradiation. In the second part of the study, predenaturation of the solid protein solder was investigated as a means for enhancing the stability of the solder in physiological fluids before irradiation. Finally, in the third part of the study, the feasibility of using synthetic polymers as a scaffold for traditional albumin protein solder mixes was investigated as a means of improving the flexibility of the solder. RESULTS: Uniform denaturation across the thickness of the solder was achieved by controlling the chromophore concentration gradient, thus ensuring stable solder-tissue fusion when the specimen was submerged in a hydrated environment. Predenaturation of the solid protein solder significantly reduced the solubility of the solder, and consequently, improved the handling characteristics of the solder. The solder-doped polymer membranes were flexible enough to be wrapped around tissue, whereas their solid nature avoided problems associated with "runaway" of the less viscous liquid solders currently used by researchers. In addition, the solder-doped polymer membranes could be easily tailored to a wide range of geometries suitable to many clinical applications. CONCLUSION: The novel solid protein solder designs presented here add a new dimension to tissue repair as their flexible, moldable, and absorption controllable nature, greatly improves the clinical applicability of laser-assisted tissue repair.

Biodegradable polymer film reinforcement of an indocyanine green-doped liquid albumin solder for laser-assisted incision closure.

BACKGROUND AND OBJECTIVE: The purpose of this study was to determine whether solid material reinforcement of a liquid albumin solder coagulum could improve the cohesive strength of the solder and, thus, the ultimate breaking strength of the incision repair in vitro. STUDY DESIGN/MATERIALS AND METHODS: A 50%(w/v) bovine serum albumin solder with 0.5 or 2.5 mg/ml indocyanine green (ICG) dye was used to repair an incision in bovine aorta. The solder was coagulated with an 806-nm continuous wave diode laser. A 50-micrometer-thick poly(DL-lactic-co-glycolic acid) film was used to reinforce the solder (the controls had solder but no reinforcement). Acute breaking strengths were measured, and the data were analyzed by Student's t-test. RESULTS: Observations of the failure modes indicate cohesive strength reinforcement of the test specimens vs. the controls. The 2.5 mg/ml ICG reinforced solder was stronger than the controls without reinforcement (P < 0.05) for all laser powers tested. There was no difference between the test specimens and the controls with 0.5 mg/ml ICG solder for low laser powers, but at higher laser powers, the reinforced solder was stronger than the controls (P < 0.05). CONCLUSION: Reinforcement of liquid albumin solders in laser-assisted incision repair seems to have advantages in terms of acute breaking strength over conventional methods that do not reinforce the cohesive strength of the solder.

Optimal parameters for laser tissue soldering: II. Premixed versus separate dye-solder techniques.

BACKGROUND AND OBJECTIVE: Laser tissue soldering by using an indocyanine green (ICG)-doped protein solder applied topically to the tissue surface and denatured with a diode laser was investigated in Part I of this study. The depth of light absorption was predominantly determined by the concentration of the ICG dye added to the solder. This study builds on that work with an in vitro investigation of the effects of limiting the zone of heat generation to the solder-tissue interface to determine whether more stable solder-tissue fusion can be achieved. STUDY DESIGN/MATERIALS AND METHODS: An alternative laser tissue soldering technique was investigated, which increased light absorption at the vital solder-tissue interface. A thin layer of ICG dye was smeared over the surface to be treated, the protein solder was then placed directly on top of the dye, and the solder was denatured with an 808-nm diode laser. Because laser light at approximately 800 nm is absorbed primarily by the ICG dye, this thin layer of ICG solution restricted the heat source to the space between the solder and the tissue surfaces. A tensile strength analysis was conducted to compare the separate dye-solder technique with conventional techniques of laser tissue soldering for which a premixed dye-solder is applied directly to the tissue surface. The effect of hydration on bond stability of repairs formed by using both techniques was also investigated using tensile strength and scanning electron microscopy analysis. RESULTS: Equivalent results in terms of tensile strength were obtained for the premixed dye-solder technique using protein solders containing 0.25 mg/ml ICG (liquid solder, 220 +/- 35 N/cm(2); solid solder, 602 +/- 32 N/cm(2)) and for the separate dye-solder technique (liquid solder, 228 +/- 41 N/cm(2); solid solder, 578 +/- 29 N/cm(2)). The tensile strength of native bovine thoracic aorta was 596 +/- 31 N/cm(2). Repairs created by using the separate dye-solder technique were more stable during hydration than their premixed dye-solder counterparts. The conventional premixed dye-solder was simpler and approximately twice as fast to apply. The separate dye-solder technique, however, increased the shelf-life of the solder, because the dye was mixed at the time of the experiment, thus conserving its spectral absorbency properties. CONCLUSION: Two laser-assisted tissue soldering techniques have been evaluated for repairing aorta incisions in vitro. The advantages and disadvantages of each of these techniques are discussed.

Behavioral sensitization after repeated formaldehyde exposure in rats.

Multiple chemical sensitivity (MCS) is a phenomenon whereby individuals report increased sensitivity to chemicals in the environment, and attribute their sensitivities to prior exposure to the same or often structurally unrelated chemicals. A leading hypothesis suggests that MCS is akin to behavioral sensitization observed in rodents after repeated exposure to drugs of abuse or environmental stressors. Sensitization occurring within limbic circuitry of the central nervous system (CNS) may explain the multisymptom complaints in individuals with MCS. The present studies represent the continuing development of an animal model for MCS, the basis of which is the CNS sensitization hypothesis. Three behaviors were assessed in rats repeatedly exposed to formaldehyde (Form) inhalation. In the first series of experiments, rats were given high-dose Form exposure (11 parts per million [ppm]; 1 h/day x 7 days) or low-dose Form exposure (1 ppm; either 1 h/day x 7 days or 1 h/day x 5 days/week x 4 weeks). Within a few days after discontinuing daily Form, cocaine-induced locomotor activity was elevated after high-dose Form or 20 days of low-dose Form inhalation. Approximately 1 month later, cocaine-induced locomotor activity remained significantly elevated in the 20-day Form-exposed rats. The second experiment assessed whether prior exposure to Form (20 days, as above) would alter the ability to condition to an odor (orange oil) paired with footshock. The results suggested a tendency to increase the conditioned fear response to the odor but not the context of the footshock box, and a decreased tendency to extinguish the conditioned fear response to odor. The third experiment examined whether CNS sensitization to daily cocaine or stress would alter subsequent avoidance responding to odor (Form). Daily cocaine significantly elevated approach responses to Form, while daily stress pretreatment produced a trend in the opposite direction, producing greater avoidance of Form. Preliminary studies indicated that repeated daily Form inhalation (20 days, as above) produced a greater avoidance to subsequent Form presentation, suggesting that daily Form inhalation may serve as a stressor. The results support the hypothesis that repeated chemical exposure in rats may produce CNS plasticity manifest as greater sensitivity to dopaminergic drugs, enhanced fear conditioning to odor paired with an aversive event, and greater avoidance of odors. Some of these behavioral changes observed in rats may provide a link with symptoms in a subset of individuals with MCS.

Optimal parameters for laser tissue soldering. Part I: tensile strength and scanning electron microscopy analysis.

BACKGROUND AND OBJECTIVES: The use of liquid and solid albumin protein solders to enhance laser tissue repairs has been shown to significantly improve postoperative results. The published results of laser-solder tissue repair studies have, however, indicated inconsistent success rates. This can be attributed to variations in laser irradiance, exposure time, solder composition, chromophore type, and concentration. An in vitro study was performed using indocyanine green-doped albumin protein solders in conjunction with an 808 nm diode laser to determine optimal laser and solder parameters for tissue repair in terms of tensile strength and stability during hydration. STUDY DESIGN/MATERIALS AND METHODS: Twenty-five different combinations of laser irradiance (6.4, 12.7, 19.1, 25.5, 31.8 W/cm2) and exposure time (20, 30, 40, 50, 100 or 40, 60, 80, 100, 200 seconds) were used. The effect of changing bovine serum albumin (BSA) concentration (25% and 60%) and indocyanine green (ICG) dye concentration (2.5 mg/ml and 0.25 mg/ml) of the protein solder on the tensile strength of the resulting bonds was investigated. The effect of hydration on bond stability was also investigated using both tensile strength and scanning electron microscopy analysis. RESULTS: Tensile strength was observed to decrease significantly with increasing irradiance. An optimum exposure time was found to exist where further irradiation did not improve the tensile strength of the bond. Tensile strength was found to be greatly improved by increasing the BSA concentration. Finally, the lower ICG dye concentration increased the penetration depth of the laser light in the protein solder leading to higher tensile strengths. The strongest repairs were formed by using 6.4 W/cm2 irradiation for 50 seconds with a protein solder composed of 60% BSA and 0.25mg/ml ICG. In addition, the solid protein solder provided more stable adhesion to the tissue than did the liquid protein solder when the tissue was submerged in a hydrated environment. CONCLUSIONS: This study greatly enhances the current understanding of the various factors affecting the soldering process. It provides a strong basis for optimization of the laser light delivery parameters and the solder constituents to achieve strong and reliable laser tissue repairs.