Effect of Electroacupuncture on Sensitive Symptoms of Distal Diabetic Peripheral Neuropathy and Its Correlation With Nerve Conduction Changes
Phase: NA
Conditions: Electroacupuncture, Acupuncture, Diabetic Polyneuropathy, Diabetic Peripheral Neuropathy, Diabetic Peripheral Neuropathic Pain, Nerve Conduction
Sponsor: Instituto Mexicano del Seguro Social

Pérez Hernández et al. 
BMC Complementary Medicine and Therapies           (2024) 24:90  
https://doi.org/10.1186/s12906-024-04375-8
STUDY PROTOCOL
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BMC Complementary
Medicine and Therapies
Electroacupuncture efficacy in diabetic 
polyneuropathy: Study protocol 
for a double‑blinded randomized controlled 
multicenter clinical trial
María Fernanda Pérez Hernández1,2,3, Alejandra Calderón Vallejo4, Sergio de Jesús Aguilar Castillo5, 
Daniel Cuauhtémoc Gómez Jiménez6, Eduardo Rodríguez Guerrero6, Fátima Aguilar Morales7, 
Macedonia Guadalupe Moreno Tovar7, Miguel Alfredo Zurita Muñóz8, Antonio Eduardo Bautista Cortéz9, 
Claudia Camelia Calzada Mendoza2, Mónica Ascención De Nova Ocampo10, 
Juan Manuel Ordóñez Rodríguez6, Mónica Luz Gómez Esquivel6, Alberto García Méndez7, Octavio Flores Gil11, 
Víctor Manuel Macías Zaragoza12, Gabriela Yanet Cortés Moreno13, Citlaltepetl Salinas Lara3,14, 
Germán Velázquez García3,12,15, Héctor Iván Saldivar Cerón3,16,17, Lucia Monserrat Pérez Navarro18, 
Laura Ávila Jiménez19, Jaime Héctor Gómez Zamudio1, Margarita Díaz Flores1, Miguel Cruz López1, 
María Esther Ocharan Hernández2 and José de Jesús Peralta Romero1,6* 
Abstract 
Background  Diabetic peripheral neuropathy (DPN) is the most common complication of type 2 diabetes mellitus 
(T2DM); its diagnosis and treatment are based on symptomatic improvement. However, as pharmacological therapy 
causes multiple adverse effects, the implementation of acupunctural techniques, such as electroacupuncture (EA) 
has been suggested as an alternative treatment. Nonetheless, there is a lack of scientific evidence, and its mecha-
nisms are still unclear. We present the design and methodology of a new clinical randomized trial, that investigates 
the effectiveness of EA for the treatment of DPN.
Methods  This study is a four-armed, randomized, controlled, multicenter clinical trial (20-week intervention period, 
plus 12 weeks of follow-up after concluding intervention). A total of 48 T2DM patients with clinical signs and symp-
toms of DPN; and electrophysiological signs in the Nerve Conduction Study (NCS); will be treated by acupuncture 
specialists in outpatient units in Mexico City. Patients will be randomized in a 1:1 ratio to one of the following four 
groups: (a) short fibre DPN with EA, (b) short fibre DPN with sham EA, (c) axonal DPN with EA and (d) axonal DPN 
with sham EA treatment. The intervention will consist of 32 sessions, 20 min each, per patient over two cycles 
of intervention of 8 weeks each and a mid-term rest period of 4 weeks. The primary outcome will be NCS parameters, 
and secondary outcomes will include DPN-related symptoms and pain by Michigan Neuropathy Screening Instru-
ment (MNSI), Michigan Diabetic Neuropathy Score (MDNS), Dolour Neuropatique Score (DN-4), Semmes-Westein 
monofilament, Numerical Rating Scale (NRS) for pain assessment, and the 36-item Short Form Health Survey (SF-36). 
*Correspondence:
José de Jesús Peralta Romero
drjperalta@hotmail.com
Full list of author information is available at the end of the article

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Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
To measure quality of life and improve oxidative stress, the inflammatory response; and genetic expression; will be 
analysed at the beginning and at the end of treatment.
Discussion  This study will be conducted to compare the efficacy of EA versus sham EA combined with conventional 
diabetic and neuropathic treatments if needed. EA may improve NCS, neuropathic pain and symptoms, oxidative 
stress, inflammatory response, and genetic expression, and it could be considered a potential coadjutant treatment 
for the management of DPN with a possible remyelinating effect.
Trial registration  ClinicalTrials.gov. NCT05521737 Registered on 30 August 2022.
International Clinical Trials Registry Platform (ICTRP) ISRCTN97391213 Registered on 26 September 2022 [2b].
Keywords  Diabetic neuropathy, Acupuncture, Electroacupuncture, Diabetes, Controlled clinical trial
Background
T2DM is a public health problem, according to the Inter-
national Diabetes Federation (IDF) it was estimated that 
537 million people had diabetes in 2021 [1, 2], which 
occurs nearly in an 80% in low-income and middle-
income countries [2], such as Mexico with an estimated 
direct cost of 50,619 million of pesos per year till 2022 
[3]. Nonetheless, only 39% of these patients have an ade-
quate glucose control, which represents a potential risk 
of developing multiple complications, such as diabetic 
neuropathy (DN) in its different types of presentation [4].
The most common type is DPN, which accounts for 
75% of DN [5]. And during the past ten years (2012–
2022), its prevalence has been reported between 7 and 
97%, worldwide [6, 7]. Although its exact epidemiologi-
cal figures are still unknown, according to a recent sys-
tematic review and meta-analysis, Mexico has the highest 
estimated prevalence, compared to other Latin American 
countries, with a prevalence close to a 69% [8].
DPN is defined as a chronic distal sensory-motor poly-
neuropathy; attributable to metabolic and microvascular 
changes caused by glycosylation phenomena, of neuronal 
proteins, microangiopathy, stimulation of autoantibodies, 
low-grade chronic inflammation, and nerve capillaries 
ischemia [9–11].
Hyperglycaemia activates autooxidation processes 
of glucose, causing nerve demyelination and stimu-
lates monocytes and endothelial cells that enhance this 
inflammatory response and damage through multiple 
signaling mechanisms that control DNA transcription; 
moreover, Nuclear factor kappa B1(NF-kB); and cytokine 
production, such as tumor necrosis factor alpha (TNF-α), 
interleukin 1β (IL-1β), interleukin 2 (IL-2), interleukin 6 
(IL-6), interleukin 18 (IL-18), and others; and release of 
reactive oxygen species (ROS), which lead to low-grade 
chronic inflammation, oxidative stress of mitochondria, 
and neuronal damage [10, 12–15].
The most common clinical manifestations of DPN are, 
neuropathic pain, hypoesthesia or anaesthesia, tingling 
sensation, and weakness, beginning in the toes symmetri-
cally [16]. However, up to 50% of all cases are asympto-
matic [17], which increases into a 25%, the risk of pelvic 
limbs amputation; due to injuries and infections predis-
position, that may go unnoticed [9, 11].
Table 1  Symptoms and clinical manifestations of DPN
Small-fiber neuropathy
Large-fiber neuropathy
Function
Nociception and protective sensation
Pressure and balance
Symptoms
Pain
Burning
Electric shocks
Stabbing
Numbness
Tingling
Poor balance
Clinical tests
Pinprick
Temperature sensation
Ankle reflexes
Vibration perception
10 g monofilament
Proprioception
Findings
Reduced or absent thermal discrimination
Reduced or absent Pinprick sensation
Reduced or absent ankle 
reflexes
Reduced or absent vibra-
tion perception
Reduced or absent 10 g 
monofilament sensation
Reduced or absent pro-
prioception

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Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
	
According to the American Diabetes Association 
(ADA), depending on symptoms and clinical examina-
tion findings [5]; DPN can be clinically classified as: 
small-fibre, large-fibre or mixed fibre polyneuropathy 
(Table 1). Altogether, this must be semi quantified with 
validated tools, such as MNSI and MDNS, which not 
only give the clinician the diagnostic suspicion of DPN; 
but can also estimate its degree of severity. Additionally, 
pain can be assessed by the DN-4 or the NRS question-
naires, to establish better strategies for pain management 
[18, 19].
Even though, clinical examination may provide an esti-
mation of nerve damage severity [20], it can only be con-
firmed by electrodiagnostic tests, such as NCS, which, 
despite of being the gold standard, this just evaluates 
large nerve fibres, whereas small fibres require other 
types of techniques. However, one of the main limitations 
of this studies is the operative and economical accessibil-
ity, since it is restricted for research purposes or differen-
tial diagnosis of atypical neuropathies [16, 18, 19, 21–23].
Due to its diagnostic difficulty, preventive measures 
have been proposed, focused mainly on glycemic control, 
modification of triggering and enhancing risk factors, 
foot care, and vitamin supplementation. But, when symp-
toms appear, the only option that is left is the instaura-
tion of pharmacological treatment with a wide variety 
of drugs, such as serotonin reuptake inhibitors, calcium 
channel blockers and tricyclic antidepressants, that, 
despite of pain relief, they can also cause multiple adverse 
effects; causing a negative impact on patients’ life qual-
ity, and a poor therapeutic adherence or abandonment of 
treatment; plus, this does not induce a nerve regenera-
tion or remyelination [5, 9, 18, 24, 25].
Therefore, nonpharmacological approaches are needed, 
and as there is evidence that glycaemic control may not 
be enough to improve neuropathic symptoms, establish-
ment of other therapies, such as, transcutaneous electri-
cal nerve stimulation, electrical spinal cord stimulation, 
or acupuncture, is recommended [9, 26].
In recent years, acupuncture; and its variants, such 
as, laser acupuncture, or EA, have gained popularity 
and have been broadly used for treating different types 
of pain. In DPN, it has demonstrated a beneficial effect 
reducing pain and neuropathic symptoms, that upgrade 
patient’s life quality; and may even induce an improve-
ment in NCS [26–29]. However, it is not yet clearly 
described whether these interventions may modify 
other responses, such as molecular or genetic profiles in 
humans, since there are few randomized controlled trials 
(RCTs) [13, 27, 30, 31].
It has been reported that EA produces an analge-
sic effect by activating cellular and neuronal regen-
eration pathways, due to the application of a small 
electric current, apart from needle insertion. Neverthe-
less, results on symptoms, electrophysiological and some 
molecular changes, are still not conclusive; because of an 
incorrect DPN stratification, a small sample size, the lack 
of a sham control or placebo group and the fulfilment of 
the ethical research guidelines requirements [32].
For this reason, we have developed a RCT, according to 
CONSORT [33] and STRICTA [34] guidelines for Good 
Clinical Practices (GCP) in medical research; guarantee-
ing a previous adequate diagnosis and DPN stratification 
by clinical and electrophysiological findings; the imple-
mentation of an intervention control group with an accu-
rate randomization, to have an stronger assessment of 
the clinical, molecular, therapeutic adherence and quality 
of life, during intervention [6a, 6b].
Objectives
The aim of this study was, to determine the efficacy, 
effectiveness, and safety of EA, on the changes of NCS 
electrophysiological parameters, in patients with DPN; 
compared to a sham EA group, during and after interven-
tion. With the assessment of other secondary objectives, 
as follows:
1.	 Semiquantitative evaluation of neuropathic symp-
toms diminishment, by the, MNSI, MDNS, DN-4 
and NRS questionnaires.
2.	 Estimation of the impact of interventions on life 
quality, by the SF-36 questionnaire.
3.	 Assessment of the modification of molecular vari-
ables, as part of pathophysiology:
a.	 Oxidative stress.
b.	 Inflammatory response.
c.	 Gene expression.
4.	 Therapeutic adherence monitoring, to establish pos-
sible interventional adverse events [7].
Trial design
This is a, four-armed, randomized, double-blinded, mul-
ticenter, placebo-controlled trial; to evaluate EA effec-
tiveness on, clinical, electrophysiological and molecular 
changes; along with its safety for DPN treatment; in an 
intention to treat (ITT) analysis, attached to real life [8].
Methods: Participants, interventions and outcomes
Study setting
This study will be conducted at the Unidad de Investi-
gación Médica en Bioquímica (UIMB), Neurology and 
Neurophysiology Departments of the Unidad Médica 
de Alta Especialidad (UMAE) “Dr. Bernardo Sepúlveda”, 

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Centro Médico Nacional (CMN) Siglo XXI, Instituto 
Mexicano del Seguro Social (IMSS), Mexico City.
With the recruitment centres at the family care units, 
UMF 20, 44 and 41, of the IMSS, Mexico City. The inter-
vention center at the Escuela Nacional de Medicina y 
Homeopatía (ENMyH), Instituto Politécnico Nacional 
(IPN), Mexico. The external collaborators for database 
management at, Facultad de Estudios Superiores (FES) 
Iztacala, Universidad Nacional Autónoma de México 
(UNAM), State of Mexico; FES Zaragoza, UNAM, Mex-
ico City; Instituto Nacional de Neurología y Neurocirugía 
“Manuel Velasco Suárez”, Mexico City; and Universidad 
Intercultural del Estado de México, Tepetlixpa, State 
of Mexico. And the statistical analysis centers at, Del-
egación Estatal de Investigación Morelos, IMSS, Cuerna-
vaca, Morelos; and Hospital General de México (HGM) 
Dr. Eduardo Liceaga, Mexico City [9].
The protocol for this trial is reported based on the 
Standard Protocol Items: Recommendations for Inter-
ventional Trials (SPIRIT) 2013 Checklist: defining stand-
ard protocol items for clinical trials (Additional files 1 
and 2).
This trial was approved on May 26th, 2020, by the Sci-
entific Research Committee IMSS (R-2020–785-070,), 
Research Ethics Committee of the Comisión Nacional 
de 
Bioética 
(CONBIOÉTICA) 
(CONBIOÉTICA: 
09CEI00920160601), and the Comisión Federal para 
la Protección Contra Riesgos Sanitarios (COFEPRIS) 
Research Committee (CI: 17 CI 09 015 006). And inter-
national trial registrations: ClinicalTrials.gov Identifier: 
NCT05521737. Registered prospectively, in August 30th, 
2022 
https://​class​ic.​clini​caltr​ials.​gov/​ct2/​show/​NCT05​
521737?​term=​elect​roacu​punct​ure&​cond=​diabe​tic+​
neuro​pathy​&​draw=​2&​rank=2 and International Clinical 
Trials Registry ISRCTN97391213. Registered on Septem-
ber 12th, 2022 – prospectively registered, https://​www.​
isrctn.​com/​ISRCT​N9739​1213 [2a].
Patients and recruitment
Sample size
According to Wang; et  al.  [31], we calculated a sample 
size with the free-use software G*Power 3.1, by a two-
sided T test for dependent samples, based on mean and 
standard deviation changes before and after treatment of 
peroneal motor nerve conduction velocity in EA group 
(40.15 ± 3.06  m/s, and 42.97 ± 2.74  m/s, respectively). 
With an 80% statistical, a 5% alpha error, and a possible 
20% loss.
Resulting on a sample size of 12 patients, per inter-
ventional group, which represents a total sample size of 
48 patients, stratified into two severity groups plus two 
interventional groups each. That is, 24 small fibres, and 
24 axonal DPN; allocated into two interventional groups 
(12 subjects for EA and 12 subjects for sham EA, per 
DPN severity group). In this context, we will recruit 
approximately 200 patients at screening, due to inclusion 
criteria [14].
Eligibility criteria
Inclusion criteria
1.	 Patients with T2DM.
2.	 Reached age between 40 and 75 years old.
3.	 Both sexes (men and women).
4.	 Reported neuropathic symptoms.
5.	 Confirmed clinical diagnosis of DPN by physical 
examination.
6.	 Electrophysiological diagnosis of DPN in its different 
types of severity (small fibres or axonal DPN).
7.	 Understanding and signing the informed consent 
form.
Exclusion criteria
1.	 Neuropathy or chronic pain caused by other condi-
tions, than diabetes.
2.	 Presence of serious medical conditions, including, 
kidney diseases, heart diseases, pulmonary diseases, 
liver diseases or contagious diseases, neurological 
diseases, treatment with cancer drugs, systemic auto-
immune diseases, hematological disorders, HIV diag-
nosis, and others.
3.	 Pregnant or breast-feeding women.
4.	 Previous history of spinal or hip trauma, fracture, or 
replacement surgery.
5.	 Ulceration of the lower extremities or previous 
amputations.
6.	 Treatment with acupuncture or moxibustion in the 
last 6 months, previous to intervention.
7.	 Patients with pacemarkers.
8.	 Unable to complete measurement questionnaires or 
physical examinations.
9.	 Recruited in other clinical trials simultaneously.
Withdrawal criteria
1.	 Development of serious adverse events during inter-
vention.
2.	 Withdrawal demanded by the participant.

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Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
	
3.	 Patients who become pregnant during interventional 
period [10].
Recruitment
To achieve the target sample size, patients will be cap-
tured at the different primary health care units, UMF 20, 
UMF 41 and UMF 44, through a telephonic invitation to 
a DPN prevention program to the potential candidates 
obtained through the databases of the recruitment cen-
tres; in which we will offer a complete neurological evalu-
ation, complemented with the electrophysiological if 
needed, as well as medical advice for prevention or con-
trol of DPN and other diabetes complications [15].
Concealment
Before randomization, a clinical physician will inform 
patients about the different procedures contemplated in 
the study, along with the possible benefits or side effects. 
After that, written consent will be obtained from each 
participant, previous to the baseline evaluation (Addi-
tional file 3) [16b, 26a].
Allocation sequence
Individual patient randomization will be performed at a 
1:1: ratio, into the four study arms of group intervention; 
using a random-numbers table by an independent statis-
tician, who will not be involved in the statistical analysis 
of the trial and will keep the allocation sequence sepa-
rately [16a].
Allocation implementation
Treatment allocation will only be revealed to acupunc-
turists out of patients’ sight, to ensure blinding, and they 
will be discouraged from discussing treatments or previ-
ous results with patients, to reduce the risk of observer 
bias [16c].
Masking
Due to the intervention characteristics, acupuncturists 
will not be blinded. However, patients and data analysis 
statisticians will be blinded to group assignment. Treat-
ment will be unrevealed to patients until all of the out-
comes and statistical analysis are completed. During the 
study, patients will receive either of the interventions 
blindfolded, placing a bandage over their eyes, so that 
they cannot see the type of therapy that will be applied 
[17a].
Procedure for unblinding if needed
Blinding will only be broken if statistically significant 
differences are obtained in the interim comparative pre-
analysis. Therefore, we might assume the need to grant 
the benefit to all patients once the study is completed in 
its entirety for all participants [17b].
Study intervention
Phases of the study
The overall Study for each patient consists of four phases, 
described below and in Fig. 1 and Additional file 4.
Phase I. Testing
Patients are captured on the database of the Primary 
Health Care Hospital, Sistema de Información de 
Medicina Familiar (SIM). After this, they are phone 
called to introduce them the protocol and make them 
the invitation to participate. If patients are interested in 
participating, a brief short form with the main eligibil-
ity criteria is applied. Once they meet the necessary eli-
gibility criteria to enter the study, an appointment at its 
family care unit is scheduled, in order to obtain the writ-
ten informed consent form, to assess the MNSI, MDNS, 
DN-4, NRS, and SF-36 questionnaires; additionally, to a 
physical neurological clinical examination by a general 
practitioner, that will establish the clinical diagnosis of 
Fig. 1  Protocol graphical abstract

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DPN. After that, the clinical diagnosis will be corrobo-
rated by a second neurological evaluation in charge of a 
neurology specialist at the neurology department of the 
UMAE “Dr. Bernardo Sepúlveda” at CMN Siglo XXI.
Phase II. Screening
For patients with a confirmed clinical and neurologi-
cal diagnosis of DPN, an electrophysiological evaluation 
by NCS will be performed; so that DPN severity can be 
assessed, and to dismiss other types of neuropathies. 
According to this, patients could be stratified into two 
main groups: short fibres and axonal DPN.
Phase III. Randomization and allocation
Once DPN severity is assessed, patients will be rand-
omized and allocated to either of the interventions (EA 
or sham EA). However, before starting the intervention, 
patients will be called to schedule a blood sample collec-
tion appointment at its UMF, for biochemical and molec-
ular test outcomes.
Phase IV. Intervention
Subsequently, patients will be contacted by acupunc-
turist technicians by phone call, to schedule their first 
interventional session. All groups will receive a total of 
32 sessions of treatment, over 37 weeks; divided into two 
cycles of intervention (EA or sham EA). That is, 16 ses-
sions each, over 8 weeks, with 2 sessions a week, and a 
rest of 4 weeks between them. Including a postinterven-
tion follow-up, extended three months after concluding 
treatment.
If patients cannot attend any of their scheduled 
appointments, they will be asked to reschedule it for 
another day in the same week.
Each patient will receive routine health care for dia-
betes and comorbidities control, during the entire 
study. Any changes in pharmacological treatment 
(including date of administration modifications, types, 
and dosage) will be recorded in detail.
At the end of each cycle of intervention, clinical, elec-
trophysiological, biochemical, and molecular param-
eters will be reassessed to compare changes during 
intervention.
Phase IV. Follow‑up
After the 32 sessions have been completed, participants 
will have a three-month additional follow-up period. 
During this term, no EA or sham EA intervention will 
be given. As well as any other type of acupuncture 
treatment will not be permitted.
Once follow-up has finished, clinical, electrophysio-
logical, biochemical, and molecular variables will be re-
evaluated to assess the intervention efficacy over time 
[13].
Interventions description
Electroacupuncture and sham EA device
Chinese HWATO-SDZ III Low-Frequency (1 to 100 Hz) 
Pulse Electronic Acupuncture Stimulator Machine, 
with Large Screen Display, 6 channels, and adjustable 
output power (0.3 to 10 V at 250 ohms). Plus 3 available 
waveforms: continuous, intermittent and both alter-
nating (Fig. 2). Both sham EA and EA devices will be 
identical in appearance, weight, sound, and operational 
procedure; but without electronic pulse stimulation 
output, for sham EA groups.
Fig. 2  Chinese HWATO-SDZ III low-frequency pulse electronic acupuncture stimulator machine

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Electroacupuncture
Patients will lie down in supine position, with their legs 
entirely exposed above the knee. The stimulated acu-
punctural points will be Zusanli (E36), Fenlong (E40), 
Yinlingquan (B9), Sanyinjiao (B6), Taichong (H3) and 
Zulinqi (VB41) [35–37] (Table 2 and Fig. 3). Acupuncture 
S-Type needles (30Gx40mm) will be inserted, with the 
application of a dense/disperse wave of 2 Hz, for 20 min 
in the different acupoints at the same time. The distance 
between the tips and the skin is approximately 1.5 and 
2 cm; until Deqi sensation is stimulated [38, 39].
Sham electroacupuncture
Sham EA will be applied using a nonpuncture device, 
made out of a Chinese Mugwort Tube Moxa Cone Self 
Stick, which prevents needle penetration; inserted at the 
same acupoints as the EA group. The device procedure 
will be identical as EA intervention, but without electrical 
stimulation, and needle manipulation, for Deqi stimula-
tion (Fig. 4) [11a].
Criteria for discontinuing modification 
of the allocated interventions
1.	 Patients demanded drop out.
2.	 Worsening or improvement of neuropathic symp-
toms.
3.	 Serious side effects that put patients’ health at risk 
[11b]
Strategies to improve adherence to interventions
For treatment adherence assessment, a specific physician 
of the UIMB, and Universidad Intercultural del Estado de 
México, Tepetlixpa, will call them once every two weeks 
during intervention term, to verify patients’ therapy 
Table 2  Acupoints
Acupoint
Location
Stimulated Nerve
Zusanli (ST36)
Located at the tibialis anterior muscle four finger breadths of subject below the kneecap and one 
finger breadth of subject lateral from the anterior crest of the tibia
Common Peroneal Nerve
Fenlong (ST40)
Eight finger breadths above the maximum prominence of the external malleolus, and 1,5 finger 
breadths laterally towards the anterior border of the tibia
Branches of Peroneal Nerve
Sanyinjiao (SP6)
Three finger breadths above the maximum protuberance of the malleolus medial, dorsally to medial 
tibial border
Tibial Nerve
Yinlingquan (SP9)
One or two finger breadths above the posteroinferior borderline of the internal tibial condyle
Saphenus Nerve
Taichong (LR3)
0.5 to 0.8 finger breadths laterally to the dorsum of the foot, in the distal depression of the angle
proximal between the 1st and 2nd metatarsal
Deep Peroneal Nerve
Zulinqi (GB41)
0.5 to 0.8 finger breadths laterally to the dorsum of the foot, at the proximal angle between the 4th 
and 5th metatarsals, in the lateral depression of the tendon of the m. extensor 5th finger longus
Branches of Tibial Nerve
Fig. 3  Acupoints location

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attendance, and to identify possible interventional side 
effects [11c].
Relevant concomitant care permitted or prohibited 
during the trial
Patients are allowed to receive any modifications for 
blood sugar, DPN pain, or chronic comorbidity control. 
Usage of complementary medicine such as traditional 
Chinese herbs or acupuncture variants; and the uptake 
of new physiotherapeutic procedures are not permitted 
[11d].
Provisions for post‑trial care
Patients who complete the whole intervention, includ-
ing the follow-up period, will receive a detailed record 
report, comparing their baseline clinical status, with the 
rest of the measurements, and medical advice to improve 
their adherence to treatment, and reduce their risk of fur-
ther complications [30].
Outcomes
All of the outcomes will be measured at the four differ-
ent timepoints (baseline, at the 2nd and 3rd visit after 
treatment cycles are completed; and at follow-up (three 
months after the end of the intervention), to evaluate 
changes with intervention (EA or sham EA).
Primary outcome
Nerve conduction study
NCS will be executed by a neurophysiologist special-
ist using the VIKING LIFE-SAVING EQUIPMENT; to 
determine the motor and sensitive NCS of the common 
peroneal, sural, and tibial nerves.
Secondary outcomes
MNSI
It will be assessed by the application of a brief dichotomic 
questionnaire of neuropathic symptoms, in which “yes” 
responses to items 1–3, 5–6, 8–9, 11–12, and 14–15, are 
each counted as one point. A “no” response on items 7 
and 13 counts as 1 point. Items 4 and 10 are not included 
in the scoring. A total score of 7 points establishes a clini-
cal diagnosis of DPN.
Then, it is completed by a physical examination, exe-
cuted by a health professional, of both lower extremities: 
foot appearance and ulceration (0 or 1 for normal and 
abnormal, respectively, ankle reflex (0 = normal, 0.5 = 
reenforced, or 1 = absent), vibration test and monofila-
ment examination (0 = normal, 0.5 = weakened, and 1= 
absent). A total score of 2.5 points will provide a clinical 
diagnosis of DPN [40].
MDNS
Measured with three main indicators, perception of 
vibration, 10 gr. monofilament and pin prick, each quali-
fied as: 0 = normal, 1 = diminished, or 2 = absent.
In addition to the muscular strength of the toes, and 
ankle dorsiflexion, quizzed as: 0 normal, 1 = diminished, 
2 = severe, or 3 = absent.
The evaluation of bicipital, tricipital, quadriceps and 
Achilles muscle reflexes, each qualified as: 0 = present, 1 
= present with effort, or 2 = absent.
A total of 0 to 6 points will be considered as no neurop-
athy, a score between 7 and 12 points as mild neuropa-
thy, 13 to 29 points as moderate neuropathy, and severe 
neuropathy if the total score is between 30 and 46 points 
[41].
Douleur Neuropathique en 4 Questions (DN‑4)
A 10-symptom item questionnaire, about the presence 
of neuropathic symptoms such as, burning, cold, electric 
shocks, tingling, numbness, itching, hypoesthesia, and 
pain. A score of 4 or more, out of 10, is suggestive of neu-
ropathic pain [18, 42].
NRS
A single 11-point numeric scale for pain assessment, 
where patients are asked to rate their current pain and 
during the past 7 days, on a scale from 0 to 10, with 0 
indicating no pain and 10 reflecting the worst possi-
ble pain of their lives. NRS is often conducted as a scale 
interpreted as follows: 0 = no pain, 1–3 = mild pain, 4–6 
= moderate pain, and 7–10 = severe pain [43].
Fig. 4  Sham electroacupuncture device

Page 9 of 14
Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
	
Quality life (SF‑36)
A validated tool for quality life measurement, that con-
sists of 36 items, divided into eight different areas: 
physical functioning, 10 items (questions 3-12); role 
limitations due to physical health, 4 items (questions 
13-16); role limitations due to emotional problems, 3 
items (questions 17-19); energy or fatigue, 4 items (ques-
tions 23, 27, 29, and 31); emotional well-being, 5 items 
(questions 24-26, 28, and 30); social functioning, 2 items 
(questions 20 and 32); pain, 2 items (questions 21 and 
22); and general health, 5 items (questions 1, 33-36). Each 
item is scored on a 0 to 100 range into a 5-grade scale, 
100, 75, 50, 25, and 0; where a higher score indicates a 
better health on each dimension. So, a 100 score repre-
sents a perfect health, while a 0 score indicates the worst 
possible health [44].
Oxidative stress
Determined by quantifying plasma concentrations of the 
lipoperoxidation subproduct; malondialdehyde (MDA), 
according to Yagi, et al. technique, where plasma MDA 
reacts with thiobarbituric acid (TBA), to form the chol-
orimetric complex “MDA-TBA”, which will be measured 
by spectrophotometry at 530 nm [45].
Inflammatory response
Changes in serum concentrations of proinflammatory 
(IL-6, IL-1β, TNF-α and IL-18) and anti-inflammatory 
(IL-10) cytokines, determined by flow cytometry quanti-
tative ELISA, with the Biolegend kit, LEGENDplex TM 
Human Inflammation Panel 1 Detection Antibodies.
Genetic expression
Measured by mRNA expression of the 5-HT1AR (seroto-
nin 1A receptor), NK-1 (neurokinin 1), α-AR1 and α-AR2 
(α-adrenoreceptors 1 and 2), NGF (nerve growth factor), 
CX3CR1 (CX3C motif chemokine receptor 1), GAP-43 
(Growth associated protein 43), NT3 (Neurotrophin), 
and CCR1 (Chemokine receptor 1) genes, quantified by 
real-time qPCR. Probes of the Human Universal Probe 
Library; and a thermal cycler Techne Prime Pro-48 Real-
Time qPCR; with a reaction mixture type TaqMan; from 
the brand "SensiFAST Probe No-ROX kit/Bioline". The 
sequences of primer oligos (sense and antisense) will be 
designed with Probe software Finder version 2.45 (http://​
www.​unive​rsalp​robel​ibrary.​com).
Safety
Adverse effect reports will be monitored during the 
entire protocol, by a specific physician responsible for 
calling patients, every two weeks to verify their health 
status, and it will be recorded in a specific format that 
will be sent to COFEPRIS annually. Just in case a major 
adverse effect is reported, such as death, permanent dam-
age, or requirement of hospitalization, it will be commu-
nicated in real-time.
Confounding variables
Anthropometry and biochemical measurements
Anthropometric variables as, height, weight and body 
mass index (BMI), waist and hip circumference; and 
blood pressure, will be measured at each timepoint 
with the Seca 213 Portable Stadiometer, Body Compo-
sition Analyser BC-418, Seca 203 measuring tape, and 
the OMRON HEM-705CPINT blood pressure monitor, 
respectively.
Biochemical parameters of fasting glucose, HbA1c%, 
total cholesterol, triglycerides, uric acid, creatinine, urea, 
and BUN; will be assessed at each timepoint with AU480 
clinical chemistry equipment; from Coulter Beckman; 
using a photometric technique to determine the concen-
trations of the analytes, which will be validated through 
the Roche Lab core System [12].
Plans for the collection, laboratory evaluation, and storage 
of biological specimens for genetic or molecular analysis 
in this trial/future use
Additionally, blood sample specimens will be collected 
to form total blood, serum, and plasma sample biobanks, 
for further molecular, genetic, and other analyses. These 
samples will be sheltered at the main research centre, 
UIMB [33].
Data collection
For the measurement of outcomes, a team will be trained 
for each of these results, that is, for neurological exami-
nation, supervised by a neurologist, for taking labora-
tories and processing biochemical and molecular tests, 
supervised by a pharmacobiological chemist, while elec-
trophysiological studies will be carried out by a neuro-
physiologist, as previously described. Data results will 
be obtained from the physical questionnaires, and elec-
trophysiological, biochemical, and molecular reports, 
which will later be digitalized and entered into an online 
platform by a clinical coordination department, in charge 
of organizing and supervising the management of this 
documents. After this, an independent researcher will 
create an online database on the same platform and cre-
ate a second digital back up on an independent computer 
owned by the UIMB and the principal investigator JJPR. 
[18a].
Data retention
All research documents, including patients’ paper files 
and electronic documents, and databases will be pre-
served for at least 7  years after the study completion 

Page 10 of 14
Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
date. Just the medical support team, the database cura-
tion supervisors and principal investigator, will have free 
access to these reports [18b].
Data management
As previously described, an independent researcher at 
UIMB, not involved in patient recruitment, intervention 
or statistical analysis; will enter each patient’s informa-
tion into a database, on a particular computer exclusively 
used for its electronic storage, apart from an online 
backup. Additionally, it will be supervised by a second 
independent researcher and third researcher at differ-
ent institutions (HGM and Delegación Estatal de Inves-
tigación Morelos, IMSS), to ensure the accuracy of the 
data [19].
Statistical analysis
Collected variables will be integrated into a Microsoft 
Access Program database; then it will be analysed in SPSS 
(Statistical Product and Service Solutions) program, ver-
sion 25, for Windows.
Descriptive analysis of collected data will be carried 
out using simple and absolute frequencies, as well as 
measures of central tendency and dispersion. Addition-
ally, to the application of the Shapiro–Wilk test, for data 
distribution assessment of quantitative variables, such as, 
MNSI, MDNS, DN-4, NRS and SF-36 total scores ques-
tionnaires, electrophysiological variables of the NCS 
(sensitive and motor latency, amplitude, and nerve veloc-
ity of sural, tibial and peroneal nerves), and biochemical 
and molecular parameters.
Changes throughout the intervention of quantitative 
variable for each of the four interventional groups at their 
different evaluation timepoints (baseline, first and sec-
ond visit, and follow-up), will be analysed by an ANOVA 
paired sample test; and to compare differences between 
interventions (EA vs sham EA), a T test for independent 
groups will be performed at each timepoint.
For the analysis of the reported symptoms of diabetic 
neuropathy during intervention, a comparison will be 
made using Cochran’s Q test for each interventional 
group; and to assess the difference in distribution of inde-
pendent groups at each study timepoint, an exact Fisher’s 
test will be applied.
Finally, multiple linear regression analysis will be per-
formed, to establish the effect size of receiving EA, 
adjusted by other independent variables that might influ-
ence the study outcomes, such as HbA1c%, and admin-
istration of pharmacological treatment for neuropathic 
pain.
For all tests, a value of p < 0.05 will be considered statis-
tically significant [20a].
Missing data analyses
The analysis will be performed with the full analysis set 
(FAS) based on an ITT analysis. Missing values will not 
be replaced. [20c].
Additional analyses
Further analyses will be regarded as explorative (with 
possible adjustment and multiple imputation for better 
results) [20b].
Interim analyses
Interim analyses are contemplated every four months, 
and just the data monitoring committee and the princi-
pal investigator, JJPR, will have access to the preliminary 
results, and he will make the final decision to finish the 
trial [21b].
Harms
Safety will be monitored by an independent clinical 
expert, with access to unblinded data, in addition to 
monthly scheduled meetings to report each patient’s clin-
ical status, and to monitor treatment efficacy and safety, 
during trial development [22].
Auditing
The principal investigator, JJPR, will prepare an annual 
report that will be delivered to the corresponding financ-
ing institutions [23].
Roles and responsibilities
Funder
Funders will only be responsible for granting financial 
support. Any changes on study design, reports, publica-
tions, or further activities are under consent of the prin-
cipal investigator, JJPR [5c].
Research committees
Medical monitoring committee
Conformed by the physicians at the recruitment and 
research centres. Who will supervise and secure the 
accomplishment of the regulatory requirements to con-
duct the protocol.
Safety review committee
An independent committee of allocation, intervention, 
data, or statistical analysis, who will monitor the clini-
cal trial progress, and review safety and effectiveness of 
interventions while the trial is still ongoing.
Data monitoring committee
It will be independent from the funders and with-
out any competing interests, conformed by MFPH at 
UIMB, LMPN at HGM “Dr. Eduardo Liceaga”, and LAJ 

Page 11 of 14
Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
	
at Delegación Estatal de Investigación Morelos, IMSS, 
Cuernavaca, in charge of doing interim comparison anal-
yses, to make recommendations on modifying, continu-
ing or finishing the trial [21a].
Clinical endpoint committee
Consisting of the data monitoring committee members, 
as well as the acupuncturists, neurologists and neuro-
physiology supervisors; and the principal investigator; 
who will review of the source data and medical events 
according to the estimated outcomes.
Steering committee
It will review the protocol, documents, or individual data, 
to provide recommendations to ensure the project suc-
cess at UIMB [5d].
Competing interests
None of the research groups received a study grant for 
conducting this clinical study, travel expenses and hono-
raria for a presentation of the scientific content, honorary 
fees for teaching acupuncture classes or lecture fees [28].
Ethics approval and consent to participate
Ethical approval was obtained on May ­26th, 2020, by 
the Scientific Research Committee (R-2020–785-070, 
IMSS), Research Ethics Committee (CONBIOÉTICA: 
09CEI00920160601), and the COFEPRIS Research Com-
mittee (CI: 17 CI 09 015 006). Trial registration: Clini-
calTrials.gov Identifier: NCT05521737. Registered 30 
August 2022—prospectively registered, https://​class​ic.​
clini​caltr​ials.​gov/​ct2/​show/​NCT05​521737?​term=​elect​
roacu​punct​ure&​cond=​diabe​tic+​neuro​pathy​&​draw=​2&​
rank=2; and ICTRP: ISRCTN97391213, registered 12 
September 2022 – prospectively registered https://​trial​
search.​who.​int/​Trial2.​aspx?​Trial​ID=​NCT05​521737.
According to the General Health Law on Health 
Research published in the Official Gazette of the Fed-
eration on February 7, 1984, this study is classified as an 
investigation with greater than the minimum risk, as it is 
a double-blind randomized clinical trial.
Its creation and development attached to the Dec-
laration of Helsinki on Ethical Principles for Medical 
Research on Human Subjects and the GCP Guidelines 
[24].
Informed consent
Therefore, the authorization of the participants through 
an informed consent form is essential. It will explain the 
objective of the study and significance, procedures, ben-
efits and potential risks, confidentiality, and the names of 
those responsible for the study and clarify the autonomy 
and freedom they must participate. In addition, as part of 
the ethical considerations, it will be reported that 20 mL 
of peripheral venous blood will be extracted, which does 
not endanger participants’ health and will be treated 
carefully according to NOM-087-ECOL-SSA1-2002, Sec-
retaría de Salud de Mexico [26b].
Confidentiality
It will be clarified that the information provided will be 
confidential, and only the medical staff and researchers 
will have access to the information. The results published 
or presented at conferences will not reveal the identity of 
the patient. All data obtained will be the property of the 
UIMB, without conflict of interest [27].
Protocol re‑approvals and amendments
Re-approvals will be made annually, as set out by the 
institution’s research and ethics committees. While 
amendments will depend on the necessary methodologi-
cal changes for the development of the protocol [25].
Consent for publication
Dissemination policy: trial results
The medical support team and acupuncturists will give 
a weekly report to the principal investigator. Principal 
investigator will submit an annual report of the adverse 
effects reported to the ethics committee and COFEPRIS. 
Participants will only receive the full report after all 
stages of the study have been completed, or in case that 
they must withdraw the study [31a].
Dissemination policy: reproducible research
Detailed information of the study is open access at the inter-
national registration platforms, clinicaltrials.gov and the 
ICTRP platform. Full access to data and statistical code will 
be considered by the principal investigator. If reviewers or 
readers have any questions regarding our published data, 
they can contact the corresponding author via email [31c].
Availability of data and materials
The principal investigator PhD José de Jesús Peralta 
Romero; has unlimited access to the final trial dataset. 
Any data needed to support the protocol can be supplied 
upon request, by the principal investigator via email at 
drjperalta@hotmail.com or drjjperalta@gmail.com [29].
Funding
At the moment, this work is supported by grants from Insti-
tuto Mexicano del Seguro Social (IMSS Prioritarios R-2020-
785-070); SECTEI (SECTEI/132/2023), and CONACHYT 
(CF-2023-G-1314) [4].

Page 12 of 14
Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
Trial status
This trial is currently recruiting patients. Until now, we 
have made two re-approvals, dated on July 9th, 2021; and 
November 18th, 2022; and we have requested a last re-
approval on January 11th, 2024. This is the second ver-
sion of the protocol, approved on the 17th of November 
2022. Recruitment began in November 2021, and the 
anticipated recruitment end date is June; 2024 [3].
Discussion
DPN is the most common complication of diabetes, caus-
ing higher rates of disability worldwide due to its diffi-
cult diagnosis and management. Even though, the main 
problem of this condition is poor glycemic control, phar-
macological treatment has a potential risk of developing 
multiple secondary events. So, it is important to establish 
different strategies for neuropathic pain and symptom 
management, such as acupuncture and its variants, such 
as EA, which have been described to benefit patients in 
pain relief, despite of glycaemic control, DPN severity, 
and other underlying patients’ conditions, with much less 
or even without any risk of presenting adverse effects, 
tolerance, dependence, addiction, or abuse as drugs.
Furthermore, it is believed that EA might have a remy-
elinating effect, as it has been reported, that, both motor 
and sensitive nerve conduction velocity, of sural and 
tibial nerves, increase significantly over time [29, 31]. 
Additionally, some authors have described in preclinical 
studies that acupoints stimulation is able to activate an 
anti-inflammatory cellular response by vagus nerve stim-
ulation, and production of acetylcholine, which inhibits 
transcription factors for proinflammatory cytokine pro-
duction [40, 46].
However, as most of them are set in animal models; and 
those that have been performed in humans, lack rand-
omization, blinding, a control group, a larger sample and 
time of intervention, further studies are needed to assess 
in a more objective manner the effect of EA.
Therefore, we chose a total intervention of 16  weeks 
with a middle-rest term of one month to prevent tachy-
phylaxis phenomena secondary to the release of mul-
tiple vasoactive mediators that may induce dangerous 
responses when administered constantly for a longer 
period [47].
On the other hand, we consider NCS changes as the 
main outcome for our study, as this is the gold stand-
ard for its highest sensitivity, so we will not only have 
the clinical diagnosis by the semiquantitative question-
naire MNSI, MDNS, DN-4, and NRS scores; we will 
stratify DPN according to electrophysiological findings. 
Additionally, other objective measures will be assessed, 
including oxidative stress, inflammation, and genetic 
expression, which is not conclusive or has not been 
well studied in humans. All of this is in consideration 
of patients’ quality of life evaluation by the SF-36 ques-
tionnaire, which makes this clinical trial a translational 
research project not only to try to elucidate EA mecha-
nisms in DPN; but also, to seek the direct application of 
the obtained results for direct patient benefit.
Abbreviations
DPN	
Diabetic polyneuropathy
T2DM	
Type 2 Diabetes Mellitus
EA	
Electroacupuncture
NCS	
Nerve Conduction Study
MNSI	
Michigan Neuropathy Screening Instrument
MDNS	
Michigan Diabetic Neuropathy Score
DN-4	
Douleur Neuropathique en 4 Questions
NRS	
Numeric Pain Rating Scale
SF-36	
36-Item Short Form Health Survey questionnaire
ICTRP	
International Clinical Trials Registry Platform
IDF	
International Diabetes Federation
DN	
Diabetic Neuropathy
DNA	
Deoxyribonucleic acid
NF-kB	
Nuclear factor kappa B1
TNF-α	
Tumor Necrosis Factor Alpha
IL-1β	
Interleukin 1β
IL-2	
Interleukin 2
IL-6	
Interleukin 6
IL-18	
Interleukin 18
ROS	
Reactive oxygen species
ADA	
American Diabetes Association
RCTs	
Randomized Controlled Trials
CONSORT	
Consolidated Standards of Reporting Trials
STRICTA​	
STandards for Reporting Interventions in Clinical Trials of 
Acupuncture
GCP	
Good Clinical Practices
ITT	
Intention to Treat
UIMB	
Unidad de Investigación Médica en Bioquímica
UMAE	
Unidad Médica de Alta Especialidad
CMN	
Centro Médico Nacional
IMSS	
Instituto Mexicano del Seguro Social
UMF	
Unidad de Medicina Familiar
ENMyH	
Escuela Nacional de Medicina y Homeopatía
IPN	
Instituto Politécnico Nacional
FES	
Facultad de Estudios Superiores
UNAM	
Universidad Nacional Autónoma de México
HGM	
Hospital General de México
SPIRIT	
Standard Protocol Items: Recommendations for Interven-
tional Trials
CONBIOÉTICA	
Comisión Nacional de Bioética
COFEPRIS	
Comisión Federal para la Protección Contra Riesgos 
Sanitarios
HIV	
Human Immunodeficiency Virus
SIM	
Sistema de Información de Medicina Familiar
MDA	
Malondialdehyde
TBA	
Thiobarbituric acid
IL-10	
Interleukin 10
ELISA	
Enzyme-linked immunosorbent assay
5-HT1AR	
Serotonin 1A receptor
NK-1	
Neurokinin 1
α-AR1	
α-Adrenoreceptor 1
α-AR2	
α-Adrenoreceptor 2
NGF	
Nerve growth factor
CX3CR1	
CX3C motif chemokine receptor 1
GAP-43	
Growth associated protein 43
NT3	
Neurotrophin
CCR1	
Chemoline receptor 1
HbA1c%	
Glycated hemoglobin

Page 13 of 14
Pérez Hernández et al. BMC Complementary Medicine and Therapies           (2024) 24:90 
	
BUN	
Blood urea nitrogen
FAS	
Full analysis set
SECTEI	
Secretaría de Educación, Ciencia, Tecnología e Innovación
CONACHYT	
Consejo Nacional de Humanidades, Ciencias y Tecnologías
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12906-​024-​04375-8.
Additional file 1. SPIRIT figure of the phases of the trial and data collec-
tion time points.
Additional file 2. SPIRIT Checklist for Trials.
Additional file 3. Informed Consent form.
Additional file 4: Supplementary figure 2. protocol consort diagram.
Acknowledgements
Unidad de Investigación Médica en Bioquímica, Unidad de Medicina Familiar 
No. 20, Unidad de Medicina Familiar No. 41, Unidad de Medicina Familiar No. 
44, Neurology and Neurophisiology Departments of the UMAE “Dr. Bernardo 
Sepúlveda”, Centro Médico Nacional Siglo XXI, IMSS; for patient recruitment. 
Escuela Nacional de Medicina y Homeopatía, FES Iztacala, FES Zaragoza, 
Universidad Intercultural del Estado de México, Tepetlixpa; for patient follow-
up. Instituto Nacional de Neurología y Neurocirugía “Manuel Velasco Suárez”, 
Delegación Estatal de Investigación Morelos, IMSS, and Hospital General de 
México “Dr. Eduardo Liceaga”, for database management; and Fundación IMSS, 
Secretaría de Educación, Ciencia Tecnología e Innovación (SECTEI), CDMX; and 
the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) 
for the financial support.
Authors’ contributions
MFPH: Conceptualization, methodology, investigation, data curation, visualiza-
tion, and writing-original draft preparation. ACV: Methodology, investigation, 
validation, resources, and writing-original draft preparation. SJAC: Methodol-
ogy, investigation, validation, resources, and writing-original draft preparation. 
DCGJ: Conceptualization, methodology, investigation, validation, resources, 
and writing-original draft preparation. ERG: Conceptualization, methodology, 
investigation, validation, resources, and writing-original draft preparation. 
FAM: Methodology, investigation, resources, and writing-review & editing 
preparation. MGMT: Methodology, investigation, resources, and writing-
review & editing preparation. MAZM: Methodology, investigation, resources, 
and writing-review & editing preparation. AEBC: Methodology, investigation, 
resources, and writing-review & editing preparation. CCCM: Methodology, 
investigation, validation, resources, and writing-review & editing preparation. 
MADNO: Methodology, investigation, validation, resources, and writing-review 
& editing preparation. JMOR: Methodology, investigation, validation, resources, 
and writing-review & editing preparation. MLGE: Methodology, investigation, 
validation, and writing-review & editing preparation. AGM: Investigation, vali-
dation, and writing-review & editing preparation. OFG: Methodology, investi-
gation, validation, resources, and writing-review & editing preparation. VMMZ: 
Methodology, investigation, validation, resources, and writing-review & editing 
preparation. GYCM: Methodology, investigation, validation, resources, and 
writing-review & editing preparation. CSL: Methodology, investigation, valida-
tion, resources, and writing-review & editing preparation. GVG: Methodology, 
investigation, validation, resources, and writing-review & editing preparation. 
HISC: Methodology, investigation, validation, resources, and writing-review & 
editing preparation. LMPN: Methodology, investigation, validation, data cura-
tion, formal analysis, and writing-review & editing preparation. LAJ: Methodol-
ogy, investigation, validation, data curation, formal analysis, and writing-review 
& editing preparation. JHGZ: Methodology, investigation, validation, resources, 
and writing-review & editing preparation. MDF: Methodology, investiga-
tion, validation, resources, and writing-review & editing preparation. MCL: 
Methodology, investigation, validation, resources, and writing-review & editing 
preparation. MEOH: Methodology, investigation, validation, resources, and 
writing-review & editing preparation. JJPR*: Conceptualization, methodology, 
investigation, validation, resources, visualization, supervision, project adminis-
tration, funding acquisition, and writing-original draft preparation.
Availability of data and materials
Data will not be openly shared, just the principal investigator PhD José de 
Jesús Peralta Romero; will have unlimited access to the final trial dataset. Any 
data needed to support the protocol can be supplied upon request, by the 
principal investigator via email at drjperalta@hotmail.com or drjjperalta@
gmail.com.
Declarations
Competing interests
The authors declare no competing interests.
Author details
1 Unidad de Investigación Médica en Bioquímica, Unidad Médica de Alta 
Especialidad “Dr. Bernardo Sepúlveda”, Centro Médico Nacional Siglo XXI, IMSS, 
Mexico City 06720, Mexico. 2 Sección de Estudios de Posgrado e Investi-
gación, Escuela Superior de Medicina, Instituto Politécnico Nacional, Mexico 
City 11340, Mexico. 3 Red MEDICI. Carrera de Médico Cirujano. FES Iztacala, 
UNAM, Estado de México, State of Mexico 54090, Mexico. 4 Departamento de 
Neurología de la Unidad Médica de Alta Especialidad ”Dr. Bernardo Sepúlveda”, 
Centro Médico Nacional Siglo XXI, IMSS, Mexico City 06720, Mexico. 5 Depar-
tamento de Neurofisiología de la Unidad Médica de Alta Especialidad “Dr. Ber-
nardo Sepúlveda”, Centro Médico Nacional Siglo XXI, IMSS, Mexico City 06720, 
Mexico. 6 Sección de Estudios de Posgrado e Investigación, Especialidad en 
Acupuntura Humana, Escuela Nacional de Medicina y Homeopatía, Instituto 
Politécnico Nacional, Mexico City 07320, Mexico. 7 Unidad de Medicina Familiar 
No. 41, IMSS, Mexico City 07760, Mexico. 8 Unidad de Medicina Familiar No. 20, 
IMSS, Mexico City 07760, Mexico. 9 Unidad de Medicina Familiar No. 44, IMSS, 
Mexico City 07320, Mexico. 10 Sección de Estudios de Posgrado e Investi-
gación, Escuela Nacional de Medicina y Homeopatía del Instituto Politécnico 
Nacional. Maestría en Ciencias en Biomedicina Molecular, Mexico City 07320, 
Mexico. 11 Coordinación de Internado y Servicio Social, Escuela Nacional de 
Medicina y Homeopatía, Instituto Politécnico Nacional, Mexico City 07320, 
Mexico. 12 Carrera de Médico Cirujano Facultad de Estudios Superiores 
Zaragoza, UNAM, Mexico City 09239, Mexico. 13 Subdirección de Servicios de 
Salud, Petróleos Mexicanos (PEMEX), Mexico City 11311, México. 14 Departa-
mento de Neuropatología, Instituto Nacional de Neurología y Neurocirugía, 
Mexico City 14269, Mexico. 15 Universidad Intercultural del Estado de México 
Plantel Tepetlixpa, Tepetlixpa, Estado de México 56880, México. 16 Carrera de 
Médico Cirujano, Facultad de Estudios Superiores Iztacala, UNAM, Tlalnepantla, 
State of Mexico 54090, Mexico. 17 Unidad de Biomedicina (UBIMED), Facultad 
de Estudios Superiores Iztacala, UNAM, Tlalnepantla, State of Mexico 54090, 
Mexico. 18 Departamento de Nefrología, Hospital General de México “Dr. 
Eduardo Liceaga”, Mexico City 06720, Mexico. 19 Coordinación Auxiliar Médica 
de Investigación en Salud, Jefatura de Servicios de Prestaciones Médicas, 
Delegación Estatal Morelos, IMSS, Cuernavaca, Morelos 62000, Mexico. 
Received: 28 December 2023   Accepted: 23 January 2024
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