Biomedical subjects

P Aebischer

Publications and source records attributed to P Aebischer.

At least 127 records · Page 7Linked to original sources

PubMed1991

Improved nerve regeneration through piezoelectric vinylidenefluoride-trifluoroethylene copolymer guidance channels.

Piezoelectric materials generating electrical charges in response to mechanical strain may be used to stimulate axonal regeneration following nerve injury. Tubular nerve guidance channels were extruded from a vinylidenefluoride-trifluoroethylene copolymer using a melt-extrusion process. Unlike vinylidenefluoride homopolymer, the copolymer does not need mechanical stretching to achieve a dipole-containing crystal structure, enabling the fabrication of complex piezoelectric devices. Selected tubes were rendered piezoelectric in a high voltage corona poling apparatus. Crystal structure changes induced by poling were evaluated with differential scanning calorimetry. In contrast to unpoled samples, poled ones displayed a sharp endothermic peak and a greater heat of transition at the Curie temperature, indicative of an increase in crystal order and size. The piezoelectric output of poled tubes was characterized using a laser-monitored deflection system interfaced with a charge amplifier and oscilloscope. Poled tubes generated significant voltages in response to slight mechanical deformations. The magnitude of electrical output was independent of the poling polarity. Unpoled tubes showed no electrical output. Positive, negative and unpoled vinylidenefluoride-trifluoroethylene copolymer tubes were used to repair a 10 mm gap in transected sciatic nerves of adult rats. Nerves regenerated in positively poled channels had a significantly greater number of myelinated axons than those regenerated in unpoled channels 4 wk post-implantation. Negatively poled channels contained an intermediate number of myelinated axons. We concluded that piezoelectrically active vinylidenefluoride-trifluoroethylene copolymer tubes significantly enhance nerve regeneration as compared to chemically identical, unpoled tubes and that the polarity of the corona poling procedure used to fabricate piezoelectric materials may play a role in determining biological responses.

Animals↗

PubMed1991

Macroencapsulation of dopamine-secreting cells by coextrusion with an organic polymer solution.

A new method of coextruding living cells in the core of a forming hollow fibre is described. PC12 cells, an immortalized cell line which secretes large amounts of dopamine, and dissociated bovine adrenal chromaffin cells, a non-dividing cell type which also secretes dopamine, were coextruded by a dry-jet wet spinning technique through a double-lumen spinneret from a 15% weight by volume solution of poly(acrylonitrile vinyl chloride) in either dimethylsulphoxide (DMSO), dimethylacetamide (DMAC) or dimethylformamide (DMF). Closure of the fibre was achieved by mounting polytetrafluoroethylene tubes on a rotating coaxial wheel system which squeezed the forming hollow fibre at regular intervals. Spontaneous and potassium-stimulated release of catecholamines from the macrocapsules were quantified under static conditions by ion-pair reverse-phase high-performance liquid chromatography equipped with electrochemical detection at 2, 4 and 6 wk. At all time periods, coextruded macrocapsules with either PC12 cells or adrenal chromaffin cells released dopamine under either unstimulated or stimulated conditions. An increase over time in dopamine release was observed from PC12 cell coextruded macrocapsules with observable difference between capsules extruded with DMSO, DMAC or DMF as solvents. Well-preserved PC12 cells and adrenal chromaffin cells were present in coextruded macrocapsules with no observable difference between capsules extruded with DMSO, DMAC or DMF as inocuity of macroencapsulation by coextrusion from an organic polymer solution. Owing to the particular fluid dynamics of this technique, minimal potentially toxic cell-solvent contact occurs allowing the use of a wider range of water-insoluble polymeric systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1991

Influence of surface texture of polymeric sheets on peripheral nerve regeneration in a two-compartment guidance system.

Synthetic guidance channels are useful tools to study the mechanisms underlying peripheral nerve regeneration. In the present study, the lumen of silicone elastomer tubes was divided into two compartments by a polymer strip 10 mm long placed along the tube length. The influence of varying the surface texture of hydrophilic and hydrophobic polymer strips on the morphology of the regenerated neural tissue was analysed. Hydrophilic nitrocellulose (NC) and hydrophobic polyvinylidene fluoride (PVDF) films with smooth (S-NC and S-PVDF) or rough (R-NC, R-PVDF) surface texture were used. Five channels of each type were used to repair transected rat sciatic nerves and analysed after 4 wk. Tissue strips bridged the nerve stumps in all R-NC and R-PVDF tubes, in five of the S-NC and three of the S-PVDF tubes. In R-NC and R-PVDF tubes, bell-shaped tissue adhering to the polymer strip was observed, whereas in S-NC and S-PVDF tubes round, free-floating nerve cables were seen. All the cables contained myelinated and unmyelinated axons and Schwann cells grouped in microfascicles and surrounded by an epineurial layer. For both rough strips, the initial cell layer consisted of macrophages adhering to the polymer surface. The epineurial nerve tissue contacting the rough surface was significantly thinner for PVDF compared with NC strips. No difference in epineurial thickness was observed for nerves facing the silicone tube or for smooth NC and PVDF strips. S-PVDF tubes contained significantly more myelinated axons than S-NC tubes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Transplantation of polymer encapsulated neurotransmitter secreting cells: effect of the encapsulation technique.

Deficits associated with neurological diseases may be improved by the transplantation within the brain lesioned target structure of polymer encapsulated cells releasing the missing neurotransmitter. Surrounding cells with a permselective membrane of appropriate molecular weight cut-off allows inward diffusion of nutrients and outward diffusion of neurotransmitters, but prevents immunoglobulins or immune cells from reaching the transplant. This technique therefore allows transplantation of post-mitotic cells across species. It also permits neural grafting of transformed cell lines since the polymer capsule prevents the formation of tumors by physically sequestering the transplanted tissue. In the present study, we compared the ability of dopamine-secreting cells, encapsulated by 2 different methods, to reverse experimental Parkinson's disease, a neurodegenerative disease characterized by motor disturbances due to a lack of dopamine within the striatum following degeneration of the dopaminergic nigro-striatal pathway. PC12 cells were loaded in polyelectrolyte-based microcapsules or thermoplastic-based macrocapsules and maintained in vitro or transplanted in a rat experimental Parkinson model for 4 weeks. Chemically-induced depolarization increased the in vitro release of dopamine from macrocapsules over time, while no increase in release was observed from microcapsules. Encapsulated PC12 cells were able to reduce lesion-induced rotational asymmetry in rats for at least 4 weeks, regardless of the encapsulation technique used. With both encapsulation methods, PC12 cell viability was greater in vivo than in vitro which suggests that the striatum releases trophic factors for PC12 cells. More brain tissue damage was observed with microcapsules than macrocapsules, possibly the result of the difficulty of manipulating the more fragile microcapsules.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Production of marked prolongation of islet xenograft survival (rat to mouse) by local release of mouse and rat antilymphocyte sera at transplant site.

Polymer rods impregnated with lyophilized particles of mouse (M) or rat (R) antilymphocyte serum (ALS) were placed adjacent to rat islet xenografts transplanted beneath the kidney capsule of diabetic mice. Insertion of rods containing only MALS or RALS had no effect on the survival time of the rat islet xenografts. In contrast, the insertion of both MALS and RALS rods with the graft produced a marked prolongation of islet xenograft survival (mean survival time greater than 55.5 +/- 10.9 days) compared with controls (14.7 +/- 2.5 days). One recipient was still normoglycemic at 100 days, and removal of the graft returned the animal to a diabetic state. The islet graft had a normal degree of beta-granulation, and a slight fibrotic reaction was present around the rods. The effect of the rods in prolonging survival of the xenografts resulted from a local slow release of MALS and RALS, because implantation of the MALS and RALS rods in the right kidney and the islets in the left kidney had no effect on prolonging islet xenograft survival. These findings indicate that local immunosuppression produced marked prolongation of rat islet xenograft survival in mice. This raises the possibility of using polymer rods for the local slow release of monoclonal antibodies to lymphokines and other agents for prevention of rejection of islet allografts and xenografts and to determine the effect of lymphokines in vivo on islet function.

Animals↗

PubMed1990

The morphology of regenerating peripheral nerves is modulated by the surface microgeometry of polymeric guidance channels.

The present study was designed to evaluate the influence of synthetic guidance channel surface microgeometry on morphological patterns of neural regeneration. Tubes with smooth (S), rough (R), or alternating smooth-rough (S/R) or rough-smooth (R/S) inner surfaces but with identical chemical composition and permeability characteristics were used to bridge a 4-mm nerve gap in a transected mouse sciatic nerve. Animals received S and R channels for 1, 2 and 4 weeks and both S/R and R/S channels for 2 and 4 weeks. At 1 week, the S tubes contained a longitudinally oriented fibrin matrix not contacting the channel's smooth inner wall, whereas R tubes featured an unorganized fibrin matrix which, together with fibroblasts and macrophages, had invaded the channel's rough trabecular network. After 4 weeks, S tubes contained discrete, free-floating nerve cables with numerous myelinated and unmyelinated axons surrounded by a thin, continuous epineurial-like layer, whereas R tubes were completely filled with a loose connective tissue stroma with only a few axons. In combined S/R or R/S channels, the general morphological patterns in individual S or R segments were similar to those observed in pure S or R channels, regardless of whether the tube segment was positioned at the proximal or distal nerve end. Proximal smooth channel segments contained discrete cables which abruptly fanned out to completely fill the lumen in distal rough segments. The opposite pattern was observed with proximal rough and distal smooth segments. At 4 weeks, myelinated axons were observed along the entire length of S/R and R/S tubes. These results suggest that the surface microgeometry of guidance channels influences the outcome of peripheral nerve regeneration, potentially by affecting the early arrangement of the fibrin matrix and/or inducing different cellular responses.

Acrylic Resins↗

PubMed1990

Effects of attachment substrates on the growth and differentiation of LLC-PK1 cells.

The growth and differentiation of an established renal epithelial cell line, LLC-PK1, on membrane bound mussel adhesive protein (MAP), collagen, and extracellular matrix (ECM) in serum-containing medium was studied. Cell attachment and growth on uncoated- vs. protein-coated cellulose nitrate and acetate membranes did not differ significantly, and confluence was achieved on all membranes. However, cells remained in a single monolayer only when plated on collagen or ECM. LLC-PK1 monolayers grown on ECM-coated membranes displayed the highest transepithelial D-glucose transport (333 +/- 22 ng.cm-2.min-1) whereas cells plated on collagen-coated membranes displayed the lowest (94 +/- 23 ng.cm-2.min-1). Glucose flux values increased with age of the culture, reaching a plateau at 28 d postseeding. These results indicate that the underlying substratum and cell age can affect differentiation of renal epithelial cells in vitro.

Aging↗

PubMed1990

NGF released from a polymer matrix prevents loss of ChAT expression in basal forebrain neurons following a fimbria-fornix lesion.

Following a unilateral fimbria-fornix lesion, the delivery of nerve growth factor (NGF) to the ipsilateral lateral ventricle of the rat can prevent the lesion-induced loss of choline acetyltransferase (ChAT) expression in the ipsilateral medial septum and vertical diagonal band region. In the present study, the ability of polymer rods to deliver NGF and to prevent a decrease in basal forebrain ChAT expression following a fimbria-fornix lesion was assessed. NGF was loaded into an ethylene vinyl acetate copolymer (EVAc) rod, fabricated by a melt-extrusion process. NGF release was established by the ability of the rods to induce neurite extension from PC12 cells and chick E12 dorsal root ganglia. Unilateral aspirative lesions of the fimbria-fornix were performed in adult rats, followed by implantation of a polymer rod into the ipsilateral lateral ventricle. Five animals received EVAc rods containing only the carrier molecule bovine serum albumin (BSA), and six received EVAc rods containing both BSA and NGF. After 2 weeks, ChAT-positive cells were counted in the medial septum and vertical diagonal band regions. Rats with NGF-releasing rods displayed ChAT(+) cell counts ipsilateral to the lesion equal to 88% of those on the contralateral side. In contrast, ChAT(+) cell numbers were 42% in animals with rods releasing BSA only (P less than 0.001). No undue reaction to implanted rods was noted. Following a fimbria-fornix lesion, NGF released from polymer matrices effectively prevents a lesion-induced reduction in ChAT expression in basal forebrain neurons.

Animals↗

PubMed1990

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗

PubMed1990

Transport functions in a bioartificial kidney under uremic conditions.

A bioartifical kidney, a confluent monolayer of renal tubular cells attached to a permselective synthetic membrane, may some day substitute for the failing renal function. In such a device, the cells would have to exhibit transport properties when exposed to uremic plasma. To test the feasibility of a bioartificial kidney, LLC-PK1 cells derived from the proximal tubule were grown to confluence on microporous, cellulose nitrate membranes and transepithelial transport of inulin, glucose and tetraethylammonium (TEA) was investigated with standard tissue culture medium and with the hemofiltrate obtained from a uremic patient. Inulin, which is neither reabsorbed nor secreted by renal epithelial cells, did not diffuse through the LLC-PK1 monolayer in either tissue culture fluid or hemofiltrate. Glucose was preferentially reabsorbed and TEA was preferentially secreted in both media. In hemofiltrate, LLC-PK1 cells reabsorbed glucose at the same rate as in tissue culture fluid but secreted TEA at a significantly higher rate. This difference was related to the pH of the fluids since it was abolished by correction to the same pH value. For both solutes, the passive transepithelial leakage was significantly lower in hemofiltrate. We conclude that LLC-PK1 cells retain their reabsorbtive and secretory transport characteristics under uremic conditions.

Animals↗

PubMed1989

Polymer electret guidance channels enhance peripheral nerve regeneration in mice.

Polytetrafluoroethylene (PTFE) tubes were prepared as electrets displaying a quasi-permanent surface charge due to the presence of trapped monopolar charge carriers. PTFE tubes containing either positive or negative charges and electrically neutral PTFE tubes were used as nerve guidance channels for the repair of a 4 mm nerve gap in the sciatic nerve of mice. After 4 weeks of implantation, positively and negatively charged PTFE electrets contained regenerated nerves with significantly more myelinated axons than nerves regenerated in uncharged PTFE tubes. This observation suggests that peripheral nerve regeneration can be enhanced by electrically charged nerve guidance channels.

Animals↗

PubMed1989

Brain tissue reaction to permselective polymer capsules.

The brain tissue reaction to permselective polymer capsules implanted in rats was evaluated for 1 to 54 weeks. The polymer capsules were well tolerated in all animals and no recognizable neurological or behavioral deficits were associated with the implants. Necrosis at the brain/polymer interface, as assessed with Nissl stain, was not observed. Foreign body giant cells were consistently absent. Immunocytochemically identified reactive neuroglial cells showed a remarkably low-grade tissue response to the synthetic material beyond the first 2 weeks of observation. Immunolabeled cortical neurons revealed conserved columnar arrays around the implants. Transmission electron microscopy showed a minimal degree of collagen deposition compared to implants in peripheral sites, and normal synapses within a few micrometers from the brain/polymer interface, supporting the prospect of biocompatible, immunoisolated xenografts in the central nervous system.

Acrylic Resins↗

PubMed1989

Tissue reaction to intraperitoneal polymer implants: species difference and effects of corticoid and doxorubicin.

The peritoneal cavity is a convenient site for implantation of encapsulated hormone-secreting tissue. However, host tissue organization around such implants may affect solute exchange and viability of the encapsulated tissue. The reaction to polyvinyl chloride acrylic copolymer capsules implanted in the peritoneal cavity of rats and mice was therefore studied. Some animals received a slow release dexamethasone pellet, others were pretreated with doxorubicin, in an attempt to minimize the tissue reaction. The tissue reaction was significantly thicker in rats than in mice at both 2 and 6 weeks after implantation. In rats, corticoids decreased significantly the thickness of the reactive layer as compared to control at all time points studied, but doxorubicin had no effect. The tissue reaction in mice was not significantly affected by corticoid treatment. In both species the thickness of the tissue reaction did not increase significantly between 2 and 6 weeks. At 3 days the tissue reaction consisted of an interrupted single layer of macrophages in mice, whereas in rats the reaction consisted of two or three layers of macrophages and polymorphonuclear cells. At both 2 and 6 weeks, several cell layers surrounded the implants: a single layer of macrophages lying along the polymer, a variable number of layers of fibroblasts interspersed with collagen fibrils (fewer in mice than in rats, and fewer in corticoid treated rats than control rats) and an outer monolayer of mesothelial cells. We conclude that the intensity of tissue reaction to polymer implants in the peritoneal cavity is species dependent and can be decreased by the administration of corticoids but not doxorubicin.

Adrenal Cortex Hormones↗

PubMed1989

Basic fibroblast growth factor released from synthetic guidance channels facilitates peripheral nerve regeneration across long nerve gaps.

Basic fibroblast growth factor (b-FGF) has been shown to enhance the in vitro survival and neurite extension of various types of neurons including dorsal root ganglia (DRG) cells. Alpha-1 glycoprotein (alpha 1-GP), an acute phase reactant, has been reported to enhance the in vitro neuritic extensions of chick DRG cells. In the present study, we investigated the ability of synthetic nerve guidance channels, which release sustained controlled amounts of b-FGF and/or alpha 1-GP, to support the regeneration of a transected peripheral nerve over a 15 mm long gap, a distance that does not permit regeneration in conventional polymeric tubes. Tubes releasing bovine serum albumin (BSA), cytochrome C, BSA and b-FGF, BSA and denatured b-FGF, BSA and alpha 1-GP, or BSA, b-FGF, and alpha 1-GP were fabricated by a dip-molding technique. In vitro kinetic studies of protein release from these channels showed an initial burst during the 1st day, followed by a linear release for at least 2 weeks thereafter. In vitro studies indicated that the b-FGF released from the polymer was biologically active as assessed by the ability of channels releasing b-FGF to induce neurite extensions in PC12 cells. For in vivo studies, the various types of tubes were used as nerve guidance channels for the repair of a 15 mm nerve gap in the sciatic nerve of rats. Four weeks postimplantation, only the tubes releasing b-FGF or b-FGF and alpha 1-GP displayed regenerated cables bridging both nerve stumps, which contained nerve fascicles with myelinated and unmyelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

An encapsulated dopamine-releasing polymer alleviates experimental parkinsonism in rats.

The effect of sustained intrastriatal release of dopamine (DA) from polymer matrices on apomorphine-induced turning behavior in a 6-hydroxydopamine (6-OHDA) unilaterally lesioned rat model was analyzed. A biocompatible semipermeable tube was placed in a denervated striatum as a receptacle for DA-releasing polymer rods. In vitro kinetics showed sustained release of DA from a polymeric rod for 15 days. Implantation of a DA-releasing rod within the striatal receptacle significantly decreased apomorphine-induced rotational behaviour in lesioned animals. Upon removal of the DA-releasing system from the receptacle, rotational behaviour increased within 2 weeks and approached preimplant control values 4 weeks later. Acute microdialysis revealed that DA appeared in the extracellular space within 20 min after the implantation of a DA-releasing rod into a denervated striatum. Significant DA amounts were still measurable 7 days postimplantation, indicating sustained DA release from the polymer rod. Dopamine released from a polymer matrix through a semipermeable receptacle alleviates experimental parkinsonism in rats, suggesting that controlled intrastriatal release of DA from a polymer matrix may provide an alternative method for the treatment of Parkinson's disease.

Acrylic Resins↗

PubMed1989

Renal epithelial-cell-controlled solute transport across permeable membranes as the foundation for a bioartificial kidney.

Despite the success of current blood purification techniques in allowing the survival of individuals with acute and chronic renal failure, the quality of life of people affected by end-stage renal disease remains unsatisfactory. Part of the reason is due to the nonphysiologic manner in which current blood purification techniques achieve homeostasis. Attempts to improve mechanical substitution of renal function by coupling the transport capability of living cells with conventional hemofiltration devices constitute the first step toward the development of an implantable bioartificial kidney.

Equipment Design↗

PubMed1989

Peripheral nerve regeneration through blind-ended semipermeable guidance channels: effect of the molecular weight cutoff.

Synthetic nerve guidance channels are used to better understand the cellular and molecular events controlling peripheral nerve regeneration. In the present study, the contribution of wound-healing molecules to peripheral nerve regeneration was assessed by varying the molecular weight cutoff of the tubular membrane. Nerve regeneration through polysulfone tubular membranes with molecular weight (Mw) cutoffs of 10(5) and 10(6) Da was analyzed in a transected hamster sciatic nerve model. Cohorts of 6 animals received tubes of either type for 4 or 8 weeks with the distal end of the polymer tube capped. Other cohorts of 6 animals received tubes of either type for 4 weeks with the distal nerve stump secured within the guidance channel so as to create a 4 or 8 mm gap between both nerve stumps. Both types of channels contained regenerated tissue cables extending to the distal end of the guidance channel at both 4 and 8 weeks in the absence of a distal nerve stump. The cables regenerated in the 10(5) Da channels were composed of nerve fascicles surrounded by a loose epineurial sheath, whereas those regenerated in the 10(6) Da channels were composed mainly of granulation tissue. The numbers of myelinated and unmyelinated axons were significantly greater in the 10(5) Da than in the 10(6) Da channels at both 4 and 8 weeks. Both types of channel contained regenerated tissue cables with numerous nerve fascicles when the distal nerve stump was present with either gap length. However, when the gap distance was 8 mm, the 10(6) Da channels contained significantly fewer myelinated axons than the 10(5) Da channels. The present study reveals that the Mw cutoff of a semipermeable guidance channel strongly influences the outcome of peripheral nerve regeneration, possibly by controlling the exchange of molecules between the channel's lumen and the external wound-healing environment. These results suggest that the wound-healing environment secretes humoral factors that can either promote or inhibit the nerve-regeneration process.

Animals↗

PubMed1988

Blind-ended semipermeable guidance channels support peripheral nerve regeneration in the absence of a distal nerve stump.

The presence of a distal nerve segment is considered to be essential for peripheral nerve regeneration through impermeable synthetic guidance channels. The use of a perm-selective material may provide a more appropriate regenerating environment by allowing solute exchange across the wall of the channel. We compared perm-selective acrylic copolymer (AC) channels with impermeable silicone elastomer (SE) channels in terms of regeneration in the absence of a distal nerve stump. Cohorts of 6 animals received AC and SE channels for either 4 or 8 weeks, with the distal end of the polymer tube left open in half of the animals, and plugged with the same polymer ('capped') in the other half. Capped and uncapped AC channels contained regenerated nerve cables which extended fully to the distal end of the channel, whereas capped SE channels contained only 1 mm long granulomatous tissue cables, and uncapped SE channels showed small cables with only a few myelinated axons. The nerve cables regenerated in uncapped AC channels were smaller and contained fewer myelinated axons than those observed in capped AC channels. Capped AC channels sleeved with a tight-fitting silicone tube to render them impermeable, showed no regenerated tissue within their lumen. The use of a perm-selective channel may have allowed the influx of nutrients and growth factors from the external environment while concentrating factors released by the proximal nerve stump.

Acrylic Resins↗