Coloured cornea replacements with anti-infective properties: expanding the safe use of silver nanoparticles in regenerative medicine.

Despite the broad anti-microbial and anti-inflammatory properties of silver nanoparticles (AgNPs), their use in bioengineered corneal replacements or bandage contact lenses has been hindered due to their intense yellow coloration. In this communication, we report the development of a new strategy to pre-stabilize and incorporate AgNPs with different colours into collagen matrices for fabrication of corneal implants and lenses, and assessed their in vitro and in vivo activity.

Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury.

Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

The involvement of SLC26 anion transporters in chloride uptake in zebrafish (Danio rerio) larvae.

After demonstrating phylogenetic relatedness to orthologous mammalian genes, tools were developed to investigate the roles of three members (A3, A4 and A6c) of the SLC26 anion exchange gene family in Cl- uptake and HCO3 excretion in embryos and larvae of zebrafish (Danio rerio). Whole-mount in situ hybridization revealed the presence of SLC26 mRNA in gill primordia, mesonephros and heart (slc26a3 and a4 only) at 5-9 days postfertilization (d.p.f.). SLC26A3 protein was highly expressed in lateral line neuromasts and within the gill, was localized to a sub-population of epithelial cells, which often (but not always) coexpressed Na+/K+-ATPase. SLC26 mRNA levels increased with developmental age, peaking at 5-10 d.p.f.; the largest increases in rates of Cl- uptake (JinCl-) preceded the mRNA spike, occurring at 2-5 d.p.f. Raising zebrafish in water with a low [Cl-] caused marked increases in JinCl- at 3-10 d.p.f. and was associated with increased levels of SLC26 mRNA. Raising fish in water of high [Cl-] was without effect on JinCl- or SLC26 transcript abundance. Selective gene knockdown using morpholino antisense oligonucleotides demonstrated a significant role for SLC26A3 in Cl- uptake in larval fish raised in control water and roles for A3, A4 and A6c in fish raised in water with low [Cl-]. Prolonged (7 days) or acute (24 h) exposure of fish to elevated (2 or 5 mmol l(-1)) ambient [HCO3-] caused marked increases in Cl- uptake when determined in water of normal [HCO3-] that were accompanied by elevated levels of SLC26 mRNA. The increases in JinCl- associated with high ambient [HCO3-] were not observed in the SLC26 morphants (significant only at 5 mmol l(-1) HCO3- for A4 and 2 mmol l(-1) HCO3- for A6c). Net base excretion was markedly inhibited in the slc26a3 and a6c morphants thereby implicating these genes in Cl-/HCO3- exchange. The results suggest that under normal conditions, Cl- uptake in zebrafish larvae is mediated by SLC26A3 Cl-/HCO3- exchangers but under conditions necessitating higher rates of high affinity Cl- uptake, SlC26A4 and SLC26A6c may assume a greater role.

Evidence that SLC26 anion transporters mediate branchial chloride uptake in adult zebrafish (Danio rerio).

Experiments were performed to test the hypothesis that three members of the SLC26 anion transporter gene family (SLC26a3, A4, and A6; hereafter termed za3, za4, and za6) mediate branchial Cl(-)/HCO(3)(-) exchange in adult zebrafish (Danio rerio). Real-time RT-PCR demonstrated that the gill expressed relatively high levels of za6 mRNA; za3 and za4 mRNA, while present, were less abundant. Also, za4 and za6 were expressed at relatively high levels in the kidney. The results of in situ hybridization or immunocytochemistry (za3 only) experiments performed on gill sections revealed that the SLC26 transporters were predominantly expressed on the filament epithelium (especially within the interlamellar regions) and to a lesser extent on the lamellar epithelium at the base of lamellae. This distribution pattern suggests that the SLC26 anion transporters are localized to mitochondrion-rich cells (ionocytes). Transferring fish to water containing low [Cl(-)] (0.02 mmol/l) resulted in significant increases in branchial SLC26 mRNA expression after 5-10 days of exposure relative to fish raised in normal water [Cl(-)] (0.4 mmol/l); transferring fish to Cl(-)-enriched water (2.0 mmol/l) was without effect on mRNA levels. Transferring fish to water containing elevated levels of NaHCO(3) (10-12.5 mmol/l) caused marked increases in branchial SLC26 mRNA expression between 3 and 10 days of transfer that was associated with a significant 40% increase in Cl(-) uptake (as measured upon return to normal water after 7 days). A decrease in whole body net acid excretion (equivalent to an increase in net base excretion) in fish previously maintained in high [NaHCO(3)] water, concurrent with increases in Cl(-) uptake and SLC26 mRNA levels, suggests a role for these anion transporters in Cl(-) uptake and acid-base regulation owing to their Cl(-)/HCO(3)(-) exchange activities.

Ventilation in Pacific hagfish (Eptatretus stoutii) during exposure to acute hypoxia or hypercapnia.

A technique was developed to measure ventilation in unrestrained Pacific hagfish (Eptatretus stoutii) by inserting and fastening into the nostril a flexible tube fitted with an ultrasonic flow probe. This technique permitted the continuous measurement of ventilation (respiratory) frequency (fR), stroke volume and minute ventilation (.V(E)) in real time in fish exposed to acute hypoxia or hypercapnia. Exposing fish to acute hypoxia (final PW(O2)=21.0 +/- 3.4 mm Hg) caused hypoxaemia and a marked increase in .V(E) of 350+/-71 ml min(-1)kg(-1) (from 235 to 585 ml min(-1)kg(-1)) owing exclusively to an increase in fR of 44+/-7 min(-1) (from 19 to 63 min(-1)). Because O(2) consumption (approximately 0.4 mmol kg(-1)h(-1)) was unaltered during hypoxia, there was an associated marked increase in the ventilation convection requirement from 36.7 to 81.8l mmol(-1). Injecting the O(2) chemoreceptor stimulant NaCN into inspired water (external CN-) or pre-branchial blood (internal CN-) evoked ventilatory responses that were similar to those observed during hypoxia although of a lesser magnitude. With external CN(-), V (E) increased maximally by 146+/-46 ml min(-1)kg(-1) and fR increased by 20+/-2 min(-1). With internal CN-, the maximal increase in .V(E) was 93+/-30 ml min(-1)kg(-1) and fR increased maximally by 19+/-6 min(-1). Exposure to acute hypercapnia (final PwC=7.0+/-0.2 mmHg) caused an increase in V (E) of 169+/-60 ml min(-1)kg(-1). These results provide compelling evidence for chemoreceptor-mediated control of breathing in hagfish and suggest that ventilatory responses to environmental hypoxia and hypercapnia in the vertebrates arose in the myxine lineage.

Chemoreceptor plasticity and respiratory acclimation in the zebrafish Danio rerio.

The goals of this study were to assess the respiratory consequences of exposing adult zebrafish Danio rerio to chronic changes in water gas composition (hypoxia, hyperoxia or hypercapnia) and to determine if any ensuing effects could be related to morphological changes in branchial chemoreceptors. To accomplish these goals, we first modified and validated an established non-invasive technique for continuous monitoring of breathing frequency and relative breathing amplitude in adult fish. Under normal conditions 20% of zebrafish exhibited an episodic breathing pattern that was composed of breathing and non-breathing (pausing/apneic) periods. The pausing frequency was reduced by acute hypoxia (Pw(O)2<130 mmHg) and increased by acute hyperoxia (Pw(O)2>300 mmHg), but was unaltered by acute hypercapnia. Fish were exposed for 28 days to hyperoxia (Pw(O)2>350 mmHg), or hypoxia (Pw(O)2=30 mmHg) or hypercapnia (Pw(CO)2=9 mmHg). Their responses to acute hypoxia or hypercapnia were then compared to the response of control fish kept for 28 days in normoxic and normocapnic water. In control fish, the ventilatory response to acute hypoxia consisted of an increase in breathing frequency while the response to acute hypercapnia was an increase in relative breathing amplitude. The stimulus promoting the hyperventilation during hypercapnia was increased Pw(CO)2 rather than decreased pH. Exposure to prolonged hyperoxia decreased the capacity of fish to increase breathing frequency during hypoxia and prevented the usual increase in breathing amplitude during acute hypercapnia. In fish previously exposed to hyperoxia, episodic breathing continued during acute hypoxia until Pw(O)2 had fallen below 70 mmHg. In fish chronically exposed to hypoxia, resting breathing frequency was significantly reduced (from 191+/-12 to 165+/-16 min(-1)); however, the ventilatory responses to hypoxia and hypercapnia were unaffected. Long-term exposure of fish to hypercapnic water did not markedly modify the breathing response to acute hypoxia and modestly blunted the response to hypercapnia. To determine whether branchial chemoreceptors were being influenced by long-term acclimation, all four groups of fish were acutely exposed to increasing doses of the O(2) chemoreceptor stimulant, sodium cyanide, dissolved in inspired water. Consistent with the blunting of the ventilatory response to hypoxia, the fish pre-exposed to hyperoxia also exhibited a blunted response to NaCN. Pre-exposure to hypoxia was without effect whereas prior exposure to hypercapnia increased the ventilatory responses to cyanide. To assess the impact of acclimation to varying gas levels on branchial O(2) chemoreceptors, the numbers of neuroepithelial cells (NECs) of the gill filament were quantified using confocal immunofluorescence microscopy. Consistent with the blunting of reflex ventilatory responses, fish exposed to chronic hyperoxia exhibited a significant decrease in the density of NECs from 36.8+/-2.8 to 22.7+/-2.3 filament(-1).

Developmental plasticity of ventilatory control in zebrafish, Danio rerio.

To determine whether development of ventilatory control in zebrafish (Danio rerio) exhibits plasticity, embryos were exposed to hypoxia, hyperoxia or hypercapnia for the first 7 days post-fertilization. Their acute reflex breathing responses to ventilatory stimuli (hypoxia, hypercapnia and external cyanide) were assessed when they had reached maturity (3 months or older). Zebrafish reared under hyperoxic conditions exhibited significantly higher breathing frequencies at rest (283+/-27min(-1) versus 212+/-16min(-1) in control fish); breathing frequency was unaffected in adult fish subjected to hyperoxia for 7 days. The respiratory responses of fish reared in hyperoxic water to acute hypoxia, hypercapnia or external cyanide were blunted (hypoxia, cyanide) or eliminated (hypercapnia). Adult fish exposed for 7 days to hyperoxia showed no change in acute responses to these stimuli. The respiratory responses to acute hypoxia, hypercapnia or external cyanide of fish reared under hypoxic or hypercapnic conditions were similar to those in fish reared under normal conditions. A subset of all fish examined exhibited episodic breathing; an analysis of breathing patterns demonstrated that fish reared under hypercapnic conditions had an increased tendency to display episodic breathing. The results of this study reveal that there is flexibility in the design and functioning of the embryonic or larval respiratory system in zebrafish.

An investigation of the role of carbonic anhydrase in aquatic and aerial gas transfer in the African lungfish Protopterus dolloi.

Experiments were performed on bimodally breathing African lungfish Protopterus dolloi to examine the effects of inhibition of extracellular vs total (extracellular and intracellular) carbonic anhydrase (CA) activity on pulmonary and branchial/cutaneous gas transfer. In contrast to previous studies on Protopterus, which showed that the vast majority of CO(2) is excreted into the water through the gill and/or skin whereas O(2) uptake largely occurs via the lung, P. dolloi appeared to use the lung for the bulk of both O(2) uptake (91.0+/-2.9%) and CO(2) excretion (76.0+/-6.6%). In support of the lung as the more important site of CO(2) transfer, aerial hypercapnia (P(CO(2))=40 mmHg) caused a significant rise in partial pressure of arterial blood CO(2) (Pa(CO(2))) whereas a similar degree of aquatic hypercapnia was without effect on Pa(CO(2)). Intravascular injection of low levels (1.2 mg kg(-1)) of the slowly permanent CA inhibitor, benzolamide, was without effect on red blood cell CA activity after 30 min, thus confirming its suitability as a short-term selective inhibitor of extracellular CA. Benzolamide treatment did not affect CO(2) excretion, blood acid-base status or any other measured variable within the 30 min measurement period. Injection of the permeant CA inhibitor acetazolamide (30 mg kg(-1)) resulted in the complete inhibition of red cell CA activity within 10 min. However, CO(2) excretion (measured for 2 h after injection) and arterial blood acid-base status (assessed for 24 h after injection) were unaffected by acetazolamide treatment. Intra-arterial injection of bovine CA (2 mg kg(-1)) caused a significant increase in overall CO(2) excretion (from 0.41+/-0.03 to 0.58+/-0.03 mmol kg(-1) h(-1)) and an increase in air breathing frequency (from 19.0+/-1.3 to 24.7+/-1.8 breaths min(-1)) that was accompanied by a slight, but significant, reduction in Pa(CO(2)) (from 21.6+/-1.6 to 19.6+/-1.8 mmHg). The findings of this study are significant because they (i) demonstrate that, unlike in other species of African lungfish that have been examined, the gill/skin is not the major route of CO(2) excretion in P. dolloi, and (ii) suggest that CO(2) excretion in Protopterus may be less reliant on carbonic anhydrase than in most other fish species.

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia.

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9+/-1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables--including heart rate (fH), blood pressure, air-breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases--were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2=46.6+/-3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9+/-2.0 to 18.8+/-6.1 nmol L(-1)) and NA (from 7.7+/-2.2 to 19.7+/-6.3 nmol L(-1)) that was accompanied by significant decreases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air-breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25+/-0.04 to 0.49+/-0.07 mmol kg(-1) h(-1)); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L(-1)), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase f(V) during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water-breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.