Fall from heights: possible factors influencing the onset of complications.

PURPOSE: Fall from a height is high energy trauma. The causes include both accidental falls and suicide attempts. The literature and also our previous study demonstrated that this kind of patients, during their recovery time, need a high multidisciplinary workload with significant costs. The present study is the first researching the patterns of the non-acute orthopedic complications after a trauma precipitation that required a new hospitalization and surgical procedure. METHODS: Retrospective study and analysis of orthopedic complication characteristics of patients fallen from height. We researched the possible relation between the complication pattern (soft tissue or bone involvement) and the case character (psychiatric or non-psychiatric patients, type of fracture and kind of fixation). RESULTS: The 18.83% of all patients (154 cases included) needed a new admission to perform further surgical procedures (9.74% of psychiatric patients and 9.09% of unvoluntary victims). Our data showed that patients with psychiatric disorder were associated with a statistically significant (p < 0.05) increase in soft tissue complications (46.67%) and onset of non-union after internal osteosynthesis and external fixation (72.72%), respectively. CONCLUSION: According to the results obtained, we can conclude that osteosynthesis in psychiatric patients is related to well-defined and predictable complications.

Comparison of Anterior and Lateral Approach in Hip Hemiarthroplasty for Femur Neck Fractures in the Elderly: Clinical and Radiographic Outcomes.

Introduction: Many surgical approaches have been described for hip hemiarthroplasty (HHA) treating femur neck fractures (FNFs). Direct lateral approach (DLA) is one of the most used. Today, the direct anterior approach (DAA) has become very attractive, but it seems to involve more intra-operative fractures. Our main endpoint was to demonstrate that the DAA may be a valid alternative comparing to the DLA. Materials and methods: Patients affected by FNFs and treated with HHA between the years 2016 and 2020 were studied. We divided the treatment of the fractures according to the surgical approach. The analysis was focused on perioperative complications and radiological outcomes. Results: There were a total of 166 patients. The DLA group included patients with an average age of 83.5 years and the DAA group of 83 years. We found similar surgical times (DLA 67 min vs DAA 61 min; p = 0,55), number of transfusions (DLA 3/person vs DAA 4/person; p = 0,91), perioperative complications (fractures: DLA 0 vs DAA 0 - dislocations: DLA 2,50% vs DAA 0) and functional outcomes (HHS: DLA 83 points vs DAA 87 points; p = 0,71). There were no statistical differences comparing diaphyseal filling (Canal Fill Index at the proximal third: DLA 0,79 vs DAA 0,78; p= 0,24), bone loss (Paprosky I: DLA 96,25% vs DAA 91,86%; p = 0,47) and prevalence of heterotopic ossification (Broker low degree: DLA 93,75% vs 95, 34%; p = 0,87). Conclusion: Analysing perioperative complications and studying post-operative radiographic evolution, our results suggest that the DAA is a valid alternative to the DLA in HHA treating FNFs.

Comparison of Anterior and Lateral Approach in Hip Hemiarthroplasty for Femur Neck Fractures in the Elderly: Clinical and Radiographic Outcomes

@#Introduction: Many surgical approaches have been described for hip hemiarthroplasty (HHA) treating femur neck fractures (FNFs). Direct lateral approach (DLA) is one of the most used. Today, the direct anterior approach (DAA) has become very attractive, but it seems to involve more intra-operative fractures. Our main endpoint was to demonstrate that the DAA may be a valid alternative comparing to the DLA. Materials and methods: Patients affected by FNFs and treated with HHA between the years 2016 and 2020 were studied. We divided the treatment of the fractures according to the surgical approach. The analysis was focused on perioperative complications and radiological outcomes. Results: There were a total of 166 patients. The DLA group included patients with an average age of 83.5 years and the DAA group of 83 years. We found similar surgical times (DLA 67 min vs DAA 61 min; p = 0,55), number of transfusions (DLA 3/person vs DAA 4/person; p = 0,91), perioperative complications (fractures: DLA 0 vs DAA 0 – dislocations: DLA 2,50% vs DAA 0) and functional outcomes (HHS: DLA 83 points vs DAA 87 points; p = 0,71). There were no statistical differences comparing diaphyseal filling (Canal Fill Index at the proximal third: DLA 0,79 vs DAA 0,78; p= 0,24), bone loss (Paprosky I: DLA 96,25% vs DAA 91,86%; p = 0,47) and prevalence of heterotopic ossification (Broker low degree: DLA 93,75% vs 95, 34%; p = 0,87). Conclusion: Analysing perioperative complications and studying post-operative radiographic evolution, our results suggest that the DAA is a valid alternative to the DLA in HHA treating FNFs.

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld(S) prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld(S) protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.

Intra-axonal calcium changes after axotomy in wild-type and slow Wallerian degeneration axons.

Calcium accumulation induces the breakdown of cytoskeleton and axonal fragmentation in the late stages of Wallerian degeneration. In the early stages there is no evidence for any long-lasting, extensive increase in intra-axonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution could have an early regulatory role in axonal degeneration in addition to the late executionary role of calcium. Schmidt-Lanterman clefts (SLCs), which allow exchange of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when axon segments are separated from the cell body, so we used the oxalate-pyroantimonate method to study calcium at SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (Wld(S)) mutant sciatic nerves, in which Wallerian degeneration is greatly delayed. In wild-type nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3mm of the lesion site by 4-24h after nerve transection. To investigate further the association with Wallerian degeneration, we studied nerves from Wld(S) rats. The step gradient of calcium distribution in Wld(S) is absent in around 20% of the intact nerves beneath SLCs but 4-24h following injury, calcium distribution in transected axons remained similar to that in uninjured nerves. We then used calcium indicators to study influx and buffering of calcium in injured neurites in primary culture. Calcium penetration and the early calcium increase in this system were indistinguishable between Wld(S) and wild-type axons. However, a significant difference was observed during the following hours, when calcium increased in wild-type neurites but not in Wld(S) neurites. We conclude that there is little relationship between calcium distribution and the early stages of Wallerian degeneration at the time points studied in vivo or in vitro but that Wld(S) neurites fail to show a later calcium rise that could be a cause or consequence of the later stages of Wallerian degeneration.

Experimental and clinical employment of end-to-side coaptation: our experience.

The last 15 years have seen a growing interest regarding a technique for nerve repair named end-to-side coaptation. Since 2000, we have carried out experimental studies on end-to-side nerve repair as well as employed this technique to a series of selected clinical cases. Here we report on the results of this experience.For experimental studies, we have used the model represented by median nerve repair by end-to-side coaptation either on the ulnar (agonistic) or the radial (antagonistic) nerve. For time course assessment of median nerve functional recovery we used the grasping test, a test which permits to assess voluntary control of muscle function. Repaired nerves were processed for resin embedding to allow nerve fibre stereology and electron microscopy. Results showed that, in either experimental group, end-to-side-repaired median nerves were repopulated by axons regenerating from ulnar and radial donor nerves, respectively. Moreover, contrary to previously published data, our results showed that voluntary motor control of the muscles innervated by the median nerve was progressively recovered also when the antagonistic radial nerve was the donor nerve.As regards our clinical experience, results were not so positive. We have treated by end-to-side coaptation patients with both sensory (n = 7, collateral digital nerves) and mixed (n = 8, plexus level) nerve lesions. Results were good, as in other series, in sensory nerves whilst they were very difficult to investigate in mixed nerves at the plexus level.Take together, these results suggest that clinical employment of end-to-side coaptation should still be considered at the moment as the ultima ratio in cases in which no other repair technique can be attempted. Yet, it is clear that more basic research is needed to explain the reasons for the different results between laboratory animal and humans and, especially, to find out how to ameliorate the outcome of end-to-side nerve repair by adequate treatment and rehabilitation.

WldS can delay Wallerian degeneration in mice when interaction with valosin-containing protein is weakened.

Axon degeneration is an early event in many neurodegenerative disorders. In some, the mechanism is related to injury-induced Wallerian degeneration, a proactive death program that can be strongly delayed by the neuroprotective slow Wallerian degeneration protein (Wld(S)) protein. Thus, it is important to understand the Wallerian degeneration mechanism and how Wld(S) blocks it. Wld(S) location is influenced by binding to valosin-containing protein (VCP), an essential protein for many cellular processes including membrane fusion and endoplasmic reticulum-associated degradation. In mice, the N-terminal 16 amino acids (N16), which mediate VCP binding, are essential for Wld(S) to protect axons, a role which another VCP binding sequence can substitute. In Drosophila, the Wld(S) phenotype is weakened by a similar N-terminal truncation and by knocking down the VCP homologue ter94. Neither null nor floxed VCP mice are viable so it is difficult to confirm the requirement for VCP binding in mammals in vivo. However, the hypothesis can be tested further by introducing a Wld(S) missense mutation, altering its affinity for VCP but minimizing the risk of disturbing other aspects of its structure or function. We introduced the R10A mutation, which weakens VCP binding in vitro, and expressed it in transgenic mice. R10AWld(S) fails to co-immunoprecipitate VCP from mouse brain, and only occasionally and faintly accumulates in nuclear foci for which VCP binding is necessary but not sufficient. Surprisingly however, axon protection remains robust and indistinguishable from that in spontaneous Wld(S) mice. We suggest that either N16 has an additional, VCP-independent function in mammals, or that the phenotype requires only weak VCP binding which may be driven forwards in vivo by the high VCP concentration.

Termino-lateral nerve suture in lesions of the digital nerves: clinical experience and literature review.

Documented experience of treatment of digital nerve lesions with the termino-lateral (end-to-side) nerve suture is limited. Our clinical experience of this technique is detailed here alongside a systematic review of the previous literature. We performed, from 2002 to 2008, seven termino-lateral sutures with epineural window opening for digital nerve lesions. Functional outcome was analysed using the two-point discrimination test and the Semmes-Weinstein monofilament test. The results showed a sensory recovery of S3+ in six cases and S3 in one case. The mean distance found in the two-point discrimination test was 12.7 mm (range 8-18 mm). After a review of the literature, we were able to obtain homogeneous data from 17 additional patients operated by termino-lateral coaptation. The overall number of cases included in our review was 24. A sensory recovery was observed in 23 out of 24 patients. The functional results were S0 in one case, S3 in one case, S3+ in twenty cases and S4 in two cases. Excluding the one unfavourable case, the mean distance in the two-point discrimination test was 9.7 mm (range 3-18 mm). It can thus be concluded that the treatment of digital nerve lesions with termino-lateral suture showed encouraging results. Based on the results obtained in this current study we believe that in case of loss of substance, end-to-side nerve coaptation may be an alternative to biological and synthetic tubulisation when a digital nerve reconstruction by means of nerve autograft is declined by the patient.

End-to-side nerve suture in traumatic injuries of brachial plexus: review of the literature and personal case series.

We used end-to-side nerve coaptation combined with standard end-to-end neurotisations to treat 11 patients who presented with complete (six cases) or incomplete (five cases) traumatic brachial plexus injuries. All patients were available for functional evaluation at a minimum of 2 years postoperatively. In three patients with shoulder abduction recovery, electromyographical studies (EMG) showed a contribution from the end-to-side neurotisation. In the remaining cases end-to-side neurotisations were unsuccessful. Our study did not demonstrate a reliable role for end-to-side nerve suture in brachial plexus surgery. We believe that at present end-to-side suture must not be a substitute for standard reconstructive techniques in brachial plexus surgery. Occasionally termino-lateral nerve sutures may represent a support to standard reconstructive procedures especially in case of severe injuries when few undamaged donor nerves are available.

A simple sterile polypropylene fingernail substitute.

Traumatic nail injuries are often observed in clinical practice. Usually the fingernail can be preserved, cleaned and disinfected in order to use it in the reconstructive procedure. However, in some cases the nail can be avulsed and lost or too damaged to be used. In cases when the nail is not available it should be replaced by a substitute in order to protect nail bed and avoid adherences along the proximal nail bed and the nail fold. Furthermore the substitute serves to protect the tender nail bed from painful stimuli during the healing process. We used, as fingernail substitute, a polypropylene sheet in eight patients with fingernail avulsion or disruption. The polypropylene foil was trimmed reproducing the profile of the avulsed fingernail and thinned at the proximal edge to reduce thickness in order to ease the insertion into the nail fold. A small hole was then created in the center of the foil to allow blood drainage. The substitute was usually removed one month after the application. In our clinical experience we had not complications related to the polypropylene device. The new fingernail had good cosmetic appearance in most cases and all the patients reported a good protection of the fingertip during the healing period. The substitute used in this series is sterile, inexpensive and easily available in emergency and elective operatory theater. This polypropylene foil is flexible and can be shaped and adapted to the nail curvature radius. The substitute used in our clinical series protected the nail bed during healing until the growth of the new fingernail and respected our functional expectations.

Alternative techniques for peripheral nerve repair: conduits and end-to-side neurorrhaphy.

Nowadays new techniques may help the surgeon in difficult cases of nerve tissue loss: when a gap is produced in a mixed nerve, the use of conduits can be an alternative to nerve grafts, which still represent the "gold standard" for this kind of lesions. We have applied biologic conduits (muscle inside a vein) in more than 40 cases since 1993 with 85% of good functional results for both sensory and mixed nerves up to 5 cm. The advantages of this technique are: (1) all graft material is easily withdrawn in the lesion area and thus is not necessary to perform any new incision; (2) the possibility of reconstructing nerve gaps up to 5 cm avoids secondary damage created by the withdrawal of healthy nerves; (3) the possibility for spontaneous orientation of regenerating nerve fibers is offered as fibers are allowed to search for their final target (chemiotropism). Furthermore, when the tissue loss is important or the proximal nerve stump is not available, so jeopardizing the possibility of recovery with traditional reconstruction, the use of end-to-side neurorrhaphy has been described to solve the problem. However the use of end-to-side neurorrhaphy in the clinical setting for motor recovery remains controversial. In our experience we had satisfying results only in 20% of cases and thus motor reconstruction in the absence of an available proximal nerve may be best handled by nerve to nerve transfers. By contrast we had good results in sensory nerve reconstruction (especially digital nerves) by end-to-side coaptation.

NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration.

The slow Wallerian degeneration protein (Wld(S)), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld(S) mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld(S) mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld(S), and lentiviral overexpressed enzyme-dead Wld(S) still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld(S) at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld(S) requires more N-terminal sequences of the protein.

Mouse Na+: HCO3- cotransporter isoform NBC-3 (kNBC-3): cloning, expression, and renal distribution.

BACKGROUND: Na+:HCO3- cotransporters mediate the transport of HCO3- into or out of the cell. We recently reported the partial cloning and characterization of a new human Na+:HCO3- cotransporter (referred to as NBC-3 or kNBC-3). The purpose of the present studies was to clone the mouse kNBC-3 and to examine its properties and expression in the kidney. METHODS: Using primers from human kNBC-3 cDNA and 5' and 3' rapid amplification cDNA end polymerase chain reaction (RACE PCR), the mouse kNBC-3 full-length cDNA was cloned from inner medullary collecting duct (mIMCD-3) cells. The tissue distribution and functional properties of NBC-3 was determined using established methods. RESULTS: The coding region of the mouse kNBC-3 has 1089 amino acids and shows 73 and 56% identity to human NBC-2 and NBC-1, respectively. The renal distribution of kNBC-3 demonstrated a unique expression pattern: Whereas kNBC-1 is predominantly expressed in the cortex and is absent in the inner medulla, kNBC-3 shows an intense expression level in the inner medulla and is absent in the cortex. Expression studies in oocytes indicated that NBC-3 mediates Na-dependent HCO3- cotransport. Electrophysiological experiments demonstrated that unlike kNBC-1, which is electrogenic, kNBC-3 is electroneutral. CONCLUSIONS: Based on its distribution and electroneutrality, we propose that kNBC-3 mediates the transport of HCO3- into the cells.

The molecular basis of O2-sensing and hypoxia tolerance in pheochromocytoma cells.

Hypoxia is a common environmental stimulus. However, very little is known about the mechanisms by which cells sense and respond to changes in oxygen. Our laboratory has utilized the PC12 cell line in order to study the biophysical and molecular response to hypoxia. The current review summarizes our results. We demonstrate that the O2-sensitive K(+) channel, Kv1.2, is present in PC12 cells and plays a critical role in the hypoxia-induced depolarization of PC12 cells. Previous studies have shown that PC12 cells secrete a variety of autocrine/paracrine factors, including dopamine, norepinephrine, and adenosine during hypoxia. We investigated the mechanisms by which adenosine modulates cell function and the effect of chronic hypoxia on this modulation. Finally, we present results identifying the mitogen- and stress-activated protein kinases (MAPKs and SAPKs) as hypoxia-regulated protein kinases. Specifically, we show that p38 and an isoform, p38gamma, are activated by hypoxia. In addition, our results demonstrate that the p42/p44 MAPK protein kinases are activated by hypoxia. We further show that p42/p44 MAPK is critical for the hypoxia-induced transactivation of endothelial PAS-domain protein 1 (EPAS1), a hypoxia-inducible transcription factor. Together, these results provide greater insight into the mechanisms by which cells sense and adapt to hypoxia.

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.

Gene expression and function of adenosine A(2A) receptor in the rat carotid body.

The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A(2A) receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A(2A) and A(2B) Ado receptor mRNAs but not the A(1) or A(3) receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A(2A) receptor mRNA. Immunohistochemical studies further showed that the A(2A) receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca(2+) currents and that this inhibition was abolished by the selective A(2A) receptor antagonist ZM-241385. Ca(2+) imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca(2+)](i). Taken together, our findings indicate that A(2A) receptors are present in type I cells and that activation of A(2A) receptors modulates Ca(2+) accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca(2+) homeostasis and cellular excitability during hypoxia.

Regulation of Shaker-type potassium channels by hypoxia. Oxygen-sensitive K+ channels in PC12 cells.

Little is known about the molecular composition of the O2-sensitive K+ (Ko2) channels. The possibility that these channels belong to the Shaker subfamily (Kv1) of voltage-dependent K+ (Kv) channels has been raised in pulmonary artery (PA) smooth muscle cells. Numerous findings suggest that the Ko2 channel in PC12 cells is a Kv1 channel, formed by the Kv1.2 alpha subunit. The Ko2 channel in PC12 cells is a slow-inactivating voltage-dependent K+ channel of 20 pS conductance. Other Kv channels, also expressed in PC12 cells, are not inhibited by hypoxia. Selective up-regulation by chronic hypoxia of the Kv1.2 alpha subunit expression correlates with an increase O2-sensitivity of the K+ current. Other Kv1 alpha subunit genes encoding slow-inactivating Kv channels, such as Kv1.3, Kv2.1, Kv3.1 and Kv3.2 are not modulated by chronic hypoxia. The Ko2 current in PC12 cells is blocked by 5 mM externally applied tetraethylammonium chloride (TEA) and by charydbotoxin (CTX). The responses of the Kv1.2 K+ channel to hypoxia have been studied in the Xenopus oocytes and compared to those of Kv2.1, also proposed as Ko2 channel in PA smooth muscle cells. Two-electrode voltage clamp experiments show that hypoxia induces inhibition of K+ current amplitude only in oocytes injected with Kv1.2 cRNA. These data indicate that Kv1.2 K+ channels are inhibited by hypoxia.