clinical prospective study on the use of subcutaneous

DDiabetic footfoot ulcer (DFU) is a serious complication of diabetes mellitus, and possibly the major mor-

bidity of the diabetic foot. It is the most common foot injury in diabetic patients and can lead to lower-

extremity amputation. Management of DFU requires a systematic knowledge of the major risk factors

for amputation, frequent routine evaluation, scrupulous preventive maintenance, and correction of periph-

eral arterial insufficiency.

Carboxytherapy refers to the subcutaneous injection of CO2 to improve the microcirculation and promote

wound-healing by stimulating the microcirculation. Since optimal ulcer-healing requires adequate tissue

perfusion, it is considered that carboxytherapy could be useful in the treatment of DFU.

The present prospective clinical study included 40 patients with different sizes and types of chronic DFU. In

addition to cleaning of the wound, antibiotics and debridement as necessary, the treatment protocol

included blood sugar control, medication, healthy habits, no weight-bearing, and carboxytherapy.

The results showed that this treatment that included carboxytherapy promoted wound-healing and

prevented amputation.

These positive effects should be confirmed through a complete study that includes different clinical and

instrumental parameters.

Clinical Prospective Study on the Use ofSubcutaneous Carboxytherapy in the

Treatment of Diabetic Foot Ulcer LYNDA KHIAT, MD

GENERAL PRACTITIONERFACULTÉ DE MÉDECINE D'ORAN

ORAN, ALGERIA

GUSTAVO H. LEIBASCHOFF, MDSPECIALIST IN OBSTETRICS AND GYNECOLOGY

FACULTY OF MEDICINE, UNIVERSITY OF BUENOS AIRESBUENOS AIRES, ARGENTINA

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#915-Leibaschoff FINAL

ABSTRACT

Advanced Wound HealingSURGICAL TECHNOLOGY INTERNATIONAL Volume 32

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Diabetic foot ulcer (DFU) is a majorcomplication of diabetes mellitus. Thewound-healing process involves foursteps: hemostasis, inflammation, prolif-eration and remodeling. The prolifera-tion step includes angiogenesis, collagendeposition, granulation, tissue formationand epithelialization. While wound-heal-ing works effectively most of the time,diabetes mellitus delays the normalphases of wound-healing.1,2 The alter-ation of the healing cascade in diabeticpatients is associated with changes in theextracellular matrix through changes infibroblasts, which lengthen and decreasein quantity. These changes in fibroblasts,in turn, lead to the reduced productionof collagen, elastin and glycosaminogly-can (GAG), and the consequent delay inthe healing and contraction of wounds.3Diabetic patients also exhibit a reducedability to generate nitric oxide from L-arginine, which compromises angiogen-esis and vasodilatation of the bloodvessels.4Uncontrolled diabetic ulcers in the

limbs are responsible for more than 25%of hospital admissions among diabeticpatients in the U.S. and U.K.5 Diabetesis the principal cause of nontraumaticlower-limb amputation in the U.S.6Everett and Mathioudakis recentlyreported that “Mortality rates associatedwith the development of a DFU are estimatedto be 5% in the first 12 months, and 5-yearmortality rates have been estimated at42%.”7In many countries with low levels of

diagnostic technology, the most impor-tant measures for avoiding foot compli-

cations in diabetic patients are carefulinspection and proper care of the diabet-ic foot. The education of diabeticpatients regarding foot hygiene, nail careand proper footwear can help reduce thepossibility of an injury that could lead tothe development of an ulcer in theextremities.8,9Adequate glycemic control, periodic

foot inspection, early recognition andproper medical treatment of these ulcerscould prevent up to 85% of amputa-tions,10,11 and the sooner a diabetic footulcer is treated, the better the result.12The risk factors for ulcer formation

include diabetic neuropathy, structuralfoot deformity and peripheral arterialocclusive disease (PAOD). PAOD is fourtimes more predominant in diabeticsthan in non-diabetics,13 and arterialocclusion typically involves the tibial andperoneal arteries. Aggravating condi-tions, like hypertension, hyperlipidemiaand smoking, also contribute to theincrease in vascular problems in diabeticpatients.14Treatment for diabetic wounds

includes both general guidelines (noweight-bearing, blood sugar control,and the promotion of healthy habits)and local treatments (e.g., removingpressure from the foot ulcer and usingtopical and general medications forwound-healing). Surgical procedures,such as skin grafts, debridement toremove dead tissue, and bypasssurgery, can be useful to restore circu-lation.Ultimately, optimal healing of diabet-

ic ulcers requires satisfactory tissue per-fusion and a healthy, oxygenated woundbed.15-17 A deficiency of oxygen (hypox-ia) in the wound bed slows or stops the

normal healing process. Hyperbaric oxy-gen therapy (HBOT) can be used as anadjunct to comprehensive wound care,however, there is some controversyregarding its effectiveness. Löndahl andco-workers 18 concluded that HBOTpromotes wound repair and enhanceshealing by improving blood circulation,encouraging angiogenesis, and providingmore oxygen to tissue in the woundbed; it also helps kill anaerobic bacteria,which cause some of the worst infec-tions in chronic wounds. On the otherhand, Fedorko and co-workers 19 wrotethat “HBOT does not offer an additionaladvantage to comprehensive wound care inreducing the indication for amputation orfacilitating wound healing in patients withchronic DFUs (diabetic foot ulcers).”Carboxytherapy is an alternative to

HBOT that consists of the therapeuticuse of carbon dioxide (CO2) in itsgaseous state via either transcutaneousapplication or subcutaneous injection.The clinical use of CO2 is not new. In

both Argentina and France, the injectionof CO2 gas has been used to treatperipheral arteriopathies, especiallyarteriopathy obliterans, in the lowerlimbs.20 When CO2 is injected subcuta-neously, it immediately diffuses at thecutaneous and muscular microcirculato-ry levels, increases microcirculatoryvasodilatation and improves flowthrough a direct action on arteriolesmooth muscle cells.21 Via the Bohreffect, it also increases the tissue PO2 atthe injection site.22-24In addition to these effects, the tis-

sue-stretching during CO2 injectioninduces slight inflammation, which trig-gers tissue repair and regeneration, andincreases macrophages, fibroblasts, and


Clinical prospective study on the use of subcutaneous carboxytherapy in the treatment of diabetic foot ulcerKHIAT/LEIBASCHOFF

INTRODUCTION

Figure 1. Carboxytherapy device. (a) side view. (b) control panel.

a b

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endothelial cells, resulting in neo-vascu-larization and remodeling of the extra-cellular matrix.25-30Based on these effects of car-

boxytherapy on the microcirculation ofthe skin, we decided to start using thistechnique in diabetic patients withulcers in their limbs. In this report, wedescribe our experience with car-boxytherapy in 40 patients with diabeticfoot ulcers.

Methods and Materials

SubjectsThis prospective clinical study includ-

ed 40 patients (13 female/27 male) withdifferent sizes and types of chronic dia-betic foot ulcers who were treatedbetween January 2015 and January 2017.The treatment protocol included

blood sugar control, medication, promo-

tion of healthy habits, no weight-bearing,and carboxytherapy. All of the patientswere volunteers from the communityfrom different regions of Algeria. They allprovided their written informed consentfor the treatment.Of the total 40 patients, 6 patients had

previously received hyperbaric oxygentherapy and 3 had received LED therapy.The remaining 31 patients had been treat-ed only with local dressings, which wasthe only treatment available in that part ofAlgeria. The average duration of car-boxytherapy was 7 months (Case 6 wastreated with CO2 for only three weeks).Healing was evaluated according to

the following criteria: a change in theodor of the dressing, the presence ofangiogenesis, new networks of blood ves-sels, changes in the exudate, a change inlocal wound color, re-epithelializationfrom the edges of the wound, andimprovement in the general condition ofthe patient (reduction of fever and betterglycemic balance).

CarboxytherapyThe CO2 device (Carboxy-pen®,

Anti-Aging Medical Systems, Montrodat,France) consists of a handpiece and a con-trol panel (Fig. 1). The electronic controlallows the user to select different para-meters, including the flow velocity, thegas volume and the dose. The CO2 itselfmust be sterile and suitable for medicaluse. CO2 is injected both systemically (at

the blue sites in Fig. 2) and locally at thewound site.Figure 3 shows an example of where

CO2 was injected in and around a wound.

Results

Clinical casesOur experiences with 6 of the 40

cases are summarized below. All of thecarboxytherapy patients were treatedwith metronidazole (500 mg, 2tablets/day) to prevent anaerobicinfection. In all cases, treatment was asfollows:75 cc or less (depending on tolerabili-

ty by the patient) of CO2 was injected ateach injection site (Fig. 2) on the internaland external sides of the right and leftlegs along the route of the large vessels ata depth of 4mm (intradermic).The same dose of CO2 was then

injected in and around the wound.



MATERIALS AND METHODS

RESULTS

Figure 2. Sites for general CO2 injection (blue).

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Case 1

A 56-year-old diabetic male underinsulin treatment presented with 2ndand 3rd degree burns on both feet withno healing over the previous 6 months.

Initial classification�Wagner Grade 2�University of Texas Grade 2 Stage B

History�Medical history = diabetes type 2 �Family history = diabetes type 2 andhigh blood pressure

�Basic Metabolic Panel = anemia

�Doppler ultrasonography of thelower extremities = plaque withweakened flow

�Radiology = no osteitis�Evolving diabetic neuropathy�Cytobacteriological study of secre-tion + antibiogram = StreptococcusB sensitive to gentamicin

Treatment�Lesions were cleaned daily with0.9% normal saline and sterile com-presses.

�Antibiotics were prescribed for 6weeks (Metronidazole 500 mg 2tablets/day and gentamicin 80 mg 1injection IM/day).

�Carboxytherapy was administeredon a schedule of 1 session every 15days for 20 sessions, for a total dura-tion of 10 months.Figures 4 and 5 show the left foot

before and after treatment (Fig. 5 (a) 1,(b) 2, (c) 4 and (d) 6 months), respective-ly. Figures 6 and 7 show the right foot

before and after treatment (Fig. 7 (a) 1,(b) 2, and (c) 4 months), respectively. Figure 8 shows both feet after 9

months of treatment.

Final classification�Wagner Grade 0�University of Texas Grade 0 Stage A



Figure 3. Example of sites for the local injection ofCO2 in and around a wound.

Figure 6. Case 1 (right foot) before treatment.

Figure 5. Case 1 (left foot) after treatment for (a)1, (b) 2, (c) 4, and (d) 6 months of treatment.

Figure 4. Photograph of Case 1 (left foot) beforetreatment.

Figure 7. Case 1 (right foot) after treatment for (a)1, (b) 2, and (c) 4 months of treatment.

Figure 8. Case 1 after treatment for 9 months.

CASE 1

a

dc

b

a

c

b

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Case 2

A 62-year-old diabetic male underinsulin treatment presented with ampu-tation of the left great toe with poorwound-healing over the previous 6months.


History�Medical history = diabetes type 2 �Surgical history = amputation of theleft great toe

�Family history = diabetes type 2 andhigh blood pressure

�Basic Metabolic Panel = anemia�Doppler ultrasonography of thelower extremities = plaque withweakened flow

�Radiology = osteitis �Evolving diabetic neuropathy�Cytobacteriological study of secre-tion+ antibiogram = Proteus sensi-tive to ciprofloxacin

Treatment�The lesion was cleaned daily with0.9% normal saline and sterile com-presses with medical honey.

�Antibiotics were administered for 8weeks (Metronidazole 500 mg 2tablets/day and ciprofloxacin 500mg 2 tablets/day)

�Carboxytherapy was administered on

a schedule of 1 session every 7 daysuntil the lesion started to improve(at 2 months), and then 1 sessionevery 15 days, for a total of 23 ses-sions over a duration of 10 months.

Figure 9 shows a photograph at theinitiation of treatment. In the photo-graph at 2 weeks (Fig. 10), osteitis isvisible. Figures 11-15 show the results after

treatment for 3, 3, 5, 6 and 8 months,respectively.




Figure 9. Photograph of Case 2 before treatment

Figure 10. Case 2 after treatment for 2 weeks.Osteitis is visible.

Figure 11. Case 2 after treatment for 3 months. Figure 12. Case 2 after treatment for 3 months.

Figure 13. Case 2 after treatment for 5 months. Figure 14. Case 2 after treatment for 6 months. Figure 15. Case 2 after treatment for 8 months.Final result with hyperkeratosis.

CASE 2

07/01/2015

20/01/2015

02/04/2015

28/05/2015 25/06/2015 03/09/2015

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CASE 3

A 54-year-old diabetic male underinsulin treatment presented withaltered wound healing at 9 months afteramputation of the left fourth toe withmany fibr in filaments and plantarinfection.


History�Medical = diabetes type 2 and highblood pressure.

�Surgical = amputation

�Family = diabetes type 2 and highblood pressure

Exam results�Basic Metabolic Panel = normal�Doppler ultrasonography of thelower extremities = plaque withweakened flow

�Radiology = No osteitis �Evolving diabetic neuropathy�ECB pus + antibiogram =Haemophilus influenza sensitive toCiprolon


�Antibiotics were prescribed for 8weeks (Metronidazole 500 mg 2tablets/day and Ciprolon 500 mg 2tablets/day)

�Carboxytherapy was administeredon a schedule of 1 session every 15days for 10 sessions, for a total dura-tion of 5 months.

Figures 16-22 show photographs atthe initiation of treatment, and aftertreatment for 1, 2, 3, 3.5, 4 and 48months, respectively.




Figure 16. Case 3 before treatment. Figure 17. Case 3 after treatment for 1month.

Figure 18. Case 3 after treatment for 2months.


Figure 20. Case 3 after treatment for3.5 months.


Figure 22. Case 3 final control photoone year after the last session of car-boxytherapy.

CASE 3

18/01/2016 16/02/2016 15/03/2016 14/04/2016

28/04/2016 November 201718/05/2016

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CASE 4

A 70-year-old diabetic male underinsulin treatment presented with alesion of the right foot that had nothealed for 3 years and showed many fib-rin filaments.Initial classification �Wagner Grade 2�University of Texas Grade 2 Stage B


�Surgical = none�Family = diabetes type 2 and high

blood pressure

Exam results�Basic Metabolic Panel = anemia�Doppler ultrasonography of thelower extremities = plaque withweakened flow and pedis stenosis

�Radiology = no osteitis �Evolving diabetic neuropathy�ECB pus+ antibiogram = Staphylo-coccus aureus sensitive to pristi-namycine


�Antibiotics were prescribed for 6weeks (Metronidazole 500 mg 2tablets/day and Pristinamycine 500mg 2 tablets/day)

�Carboxytherapy was administered ona schedule of 1 session every 15 daysfor 16 sessions, for a total durationof 8 months.

Figures 23-27 show photographs atthe initiation of treatment, and aftertreatment for 1, 2, 3, and 6 months,respectively.




Figure 23. Case 4 before treatment. Figure 24. Case 4 after treatment for 1 month. Figure 25. Case 4 after treatment for 2 months.

Figure 26. Case 4 after treatment for 3 months. Figure 27. Case 4 after treatment for 6 months.

CASE 4

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CASE 5

A 53-year-old diabetic male underinsulin treatment presented with awound in the left foot that had nothealed for 8 months and which showedlocalized necrosis.

Initial classification�Wagner Grade 4�University of Texas Grade 3 Stage D


�Surgical = none

�Family = diabetes type 2 and highblood pressure

Exam results�Basic Metabolic Panel = anemia�Doppler ultrasonography of thelower extremities = plaque withweakened flow

�Radiology = osteitis �Evolving diabetic neuropathy�ECB pus+ antibiogram = Klebsiellasensitive to ciprofloxacin

Treatment�The lesion was cleaned daily with0.9% normal saline and sterile com-presses with medical honey.

�Antibiotics were administered for 8

weeks (Metronidazole 500 mg 2tablets/day and ciprofloxacin 500mg 2 tablets/day)

�Carboxytherapy was administeredon a schedule of 1 session every 15days, for a total of 15 sessions over aduration of 7.5 months.

Figures 28-34 show photographs atthe initiation of treatment, and aftertreatment for 1, 2, 3, 4, 7 and 48months, respectively.




Figure 28 Case 5 before treatment. Figure 29. Case 5 after treatment for 1 month. Figure 30. Case 5 after treatment for 2 months.

Figure 31. Case 5 after treatment for3 months.



Figure 34. Case 5 final control photoone year after the last session of car-boxytherapy (November 2017).

CASE 5

November 2017

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CASE 6

A 54-year-old diabetic female underoral antidiabetic treatment presentedwith a lesion on her left foot that hadnot healed for 6 months.


History�Medical = diabetes type 2�Surgical = appendectomy�Family = diabetes type 2 and high

blood pressure Exam results�Basic Metabolic Panel =normal �Doppler ultrasonography of thelower extremities = plaque withweakened flow

�Radiology = no osteitis�Evolving diabetic neuropathy�Cytobacteriological study of secre-tion + antibiogram = Staphylococ-cus aureus sensitive to gentamicin

Treatment�The lesion was cleaned daily with0.9% normal saline and sterile com-presses.

�Antibiotics were administered for 3weeks (Metronidazole 500 mg 2tablets/day and gentamicin 80mg 1injection IM/day)

�Carboxytherapy was administered ona schedule of 1 session every 7 daysfor 2 sessions.

Figures 35-37 show photographs atthe initiation of treatment, and aftertreatment for 1 and 3 weeks, respec-tively.




Figure 35. Case 6 before treatment.

Figure 36. Case 6 after treatment for 1 week. Figure 37. Case 6 after treatment for 3 weeks.

CASE 6

Discussion

Delayed healing and chronic woundsare some of the basic characteristics ofdiabetic patients. Wounds in theextremities of diabetic patients thatrequire long-term treatment affect allareas of the patient’s life and lead to areduced quality of life. For example,diabetic foot ulceration is a major pre-condition to amputation; 85% of dia-betic patients undergoing amputationhave had a prior foot ulceration.31

Microcirculation in diabetic ulcers inthe limbs is significantly different fromthat in healthy skin, and can lead tolocal hypoxia, edema, microaneurysmsand microhemorrhages, as observed byvideo capillaroscopy. Revascularizationis the key to treatment for ischemic dia-betic lesions.32 Various medical, physio-therapy and surgical techniques havebeen developed to improve the micro-circulation and thus avoid negativeeffects on tissue tropism. In this clinicalstudy, in addition to basic treatment of

the lesion (cleaning, antibiotic treat-ment, correction of support), patientswith diabetic foot ulcers were treatedby the subcutaneous injection of CO2.At the vascular level, CO2 increases vas-cular tone and produces active micro-circulatory vasodilatation. CO2-inducedvasodilatation results from its directactivity on arteriole smooth musclecells.21In addition, CO2 promotes Bohr’s

effect, a mechanism that allows thetransfer of tissue CO2 to the lungs and

DISCUSSION

the transfer of lung O2 to tissuesthrough the oxy-hemoglobin dissocia-tion curve. When administered throughan external route, CO2 promotes thismechanism, resulting in a higher tissueoxygenation and neoangiogenesis.Of the 40 patients in this study, none

required amputation. Furthermore, 31showed complete recovery and 9showed partial recovery.

Conclusion

Based on the present observations,carboxytherapy appears to be a validtreatment option in diabetic patientswith microcirculatory disturbances thatproduce trophic injury. While furtherstudies will be needed to compare theresults with carboxytherapy to thosewith other techniques, none of the 40patients we treated required amputationand all made a full functional recovery.The results of this prospective clini-

cal study may help to improve the treat-ment of diabetic patients with impairedwound-healing in their extremities andconsequently improve their quality oflife.

Authors Disclose

The authors have no conflicts ofinterest to disclose.

References

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