Neuroaxonal Injury May Mediate the Association Between Hyperglycemia and Prognosis in Spontaneous Subarachnoid Hemorrhage

Vol.:(0123456789) Molecula Neu obiology (2025) 62:1467–1477 h ps://doi.o g/10.1007/s12035-024-04347-6 ORIGINAL ARTICLE Neu oaxonal Inju y May Media e heAssocia ion Be ween Hype glycemia andP ognosis inSpon aneous Suba achnoid Hemo hage DanielSan ana1 · Lau aLlull1 · Alejand aMos ei o2 · Lei ePed osa3 · Gab ielPujol4 · LuigiZa e a5 · Ma ianoWe ne 6 · Ab ahamMa ín7,8 · Ca lesJus icia9 · ÁngelChamo o1 · RamónTo né2 · Se gioAma o1 Recei ed: 4 Ap il 2023 / Accep ed: 2 July 2024 / Published online: 12 July 2024 © The Au ho (s) 2024 Abs ac Hype glycemia du ing ea ly b ain inju y (EBI) pe iod a e spon aneous suba achnoid hemo hage (SAH) is associa ed wi h poo ou come, bu he unde lying physiopa hology is unknown. This s udy assessed i hype glycemia du ing EBI is associa ed wi h ma ke s o neu oaxonal inju y and whe he hese bioma ke s pa ially accoun o he associa ion be ween hype glycemia and poo clinical ou come. Nine y- wo SAH pa ien s admi ed wi hin 24h o bleeding onse we e p ospec i ely included. Glucose le els we e measu ed a a i al and e e y 6h o 72h. Se um neu o ilamen ligh chain (NFL) le els we e meas- u ed a 72h. Func ional ou come was assessed wi h he modi ied Rankin Scale (mRS) a 90days (poo ou come, mRS > 2). The associa ion be ween glucose me ics, NFL le els, and clinical ou come was assessed wi h uni a ia e and mul i a ia e analyses. Media ion analysis was pe o med o examine he po en ial chain in which NFL may media e he ela ionship be ween glucose and unc ional ou come. Highe glucose and NFL le els du ing EBI we e associa ed wi h poo clinical ou come in adjus ed analysis. NFL le els we e associa ed wi h olde age, highe ini ial se e i y, and highe glucose le els du ing EBI pe iod. In adjus ed media ion analyses, he associa ion be ween glucose and clinical ou come was signi ican ly media ed by NFL le els. The media o NFL explained 25% o he associa ion be ween glucose du ing EBI pe iod and poo unc ional ou come a 90days. In SAH, he associa ion be ween glucose le els du ing EBI and poo clinical ou come migh be signi ican ly media ed by NFL le els. The link be ween hype glycemia and poo clinical ou come migh be explained in pa h ough seconda y neu oaxonal inju y. Keywo ds Suba achnoid hemo hage· Ea ly b ain inju y· Neu o ilamen · Glucose· Neu oaxonal damage· Media ion analysis * Ramón To né [email p o ec ed] * Se gio Ama o [email p o ec ed] 1 Ins i u e o Neu oscience, Comp ehensi e S oke Cen e , Hospi al Clinic o Ba celona; Uni e si y o Ba celona, Ba celona, Spain 2 Ins i u e o Neu oscience, Neu osu ge y Depa men , Hospi al Clinic o Ba celona; Uni e si y o Ba celona, Ba celona, Spain 3 Ins i u d’In es igacions Biomèdiques Agus í Pi I Sunye (IDIBAPS), Ba celona, Spain 4 Neu oanes hesia Di ision, Anes hesiology Depa men , Hospi al Clinic o Ba celona, Ba celona, Spain 5 Neu oc i ical Ca e Di ision, An hesiology Depa men , Hospi al Clinic o Ba celona, Ba celona, Spain 6 Ins i u e o Diagnos ic Imaging, In e en ional Neu o adiology Depa men , Hospi al Clinic o Ba celona, Ba celona, Spain 7 Achuca o Basque Cen e o Neu oscience, Leioa, Spain 8 Ike basque Basque Founda ion o Science, Bilbao, Spain 9 Ins i u o de In es igaciones Biomédicas de Ba celona (IIBB), Consejo Supe io de In es igaciones Cien í icas (CSIC), Ba celona, Spain 1468 Molecula Neu obiology (2025) 62:1467–1477 In oduc ion Spon aneous suba achnoid hemo hage (SAH) is a de as- a ing ce eb o ascula disease caused by he up u e o an in ac anial aneu ysm in 85% o cases [1, 2]. The disabili y bu den caused by SAH is pa ly d i en by se e al ha m ul mechanisms igge ed wi hin he i s 72h a e bleeding onse esul ing in he so-called ea ly b ain inju y (EBI), which has been consis en ly associa ed wi h an inc eased isk o delayed sys emic complica ions as well as long- e m poo ou come [3, 4]. The mechanisms implica ed in EBI a e s ill pa ly known and include mic o h ombosis, oxida i e s ess, blood–b ain ba ie dis up ion, and mul i ocal axonal inju y, among o he s [5–10]. In acu e cen al ne ous sys- em diseases including b ain hemo hage, mos o hese dele e ious mechanisms a e enhanced a e he exposi ion o high glucose le els [11–14]. Hype glycemia occu s in abou h ee ou o ou SAH pa ien s wi hin he EBI pe iod, and i has been associa ed wi h poo clinical ou - come [15–20]. Hype glycemia is in pa a consequence o an acu e s ess esponse o b ain inju y and enhances a my iad o dele e ious physiopa hological p ocesses includ- ing neu oin lamma o y mechanisms ha e en ually may p omo e di use neu oaxonal damage (NAD) [21–23]. Howe e , he e a e no epo s on he link be ween abno - mal glucose p o iles du ing he EBI pe iod and NAD bioma ke s. Neu o ilamen ligh chains (NFL) a e s uc u al p o eins mainly p esen in he cy oskele on o myelina ed neu ons. Following axonal inju y, NFLs a e eleased o he ex a- cellula compa men hence being conside ed su oga e bioma ke s o NAD [24]. In he se ing o SAH, NFL le els measu ed in blood and ce eb ospinal luid samples collec ed du ing he EBI pe iod as bioma ke s o ongoing b ain inju y a e s ongly co ela ed wi h poo clinical ou - come me ics a sho and long e m [25–33]. These obse - a ions sugges ha NAD could play a majo ole in he EBI phase a e SAH, al hough he mechanisms implica ed in he se e i y o NAD emain o be ully unde s ood. He ein, we hypo hesized ha in SAH su e e s, he p ognos ic ele ance o hype glycemia du ing he EBI pe iod could be a leas in pa media ed h ough i s ela- ionship wi h NAD. Thus, ou speci ic objec i e was o e alua e in a p ospec i e coho o SAH subjec s whe he he associa ion be ween hype glycemia and poo clinical ou come was media ed by di use NAD as measu ed wi h ci cula ing NFL le els a he end o he EBI pe iod. E en- ually, de e mining he link be ween glucose dys egula- ions du ing he EBI pe iod, NAD and long- e m clinical ou come could be o in e es o designing and moni o ing he e ec s o new in e en ions in clinical ials aimed o modula e glucose le els o he mechanisms associa ed wi h glucose-d i en seconda y b ain inju y. Me hods S udy Design andPopula ion We conduc ed an obse a ional, single-cen e , p ospec i e coho s udy in a e ia y le el e e al hospi al (> 50 SAH admissions pe yea ) p o ided wi h a mul idisciplina y Com- p ehensi e S oke Cen e , a S oke Uni and an In ensi e Ca e Uni specialized in neu oc i ical ca e. Pa icipan s we e included in he s udy om Sep embe 2018 o June 2021. The s udy inclusion c i e ia we e age olde han 18yea s, good p emo bid unc ional s a us [modi ied Rankin Scale (mRS) 0–2], and spon aneous SAH admi ed a ou cen e in he i s 24h om symp om onse . Exclusion c i e ia included he p esence o a non-aneu ysmal exclusi ely pe - imesencephalic blood pa e n in he admission CT, p e ious his o y o diabe es melli us, SAH seconda y o o he causes (i.e., auma ic, enous h ombosis, myco ic aneu ysms), o e y sho li e expec ancy ha could comp omise ollow- up. A schema ic lowcha o he s udy is shown in Supple- men a y Fig.1. E hics app o al was ob ained om he local Clinical Resea ch E hics Commi ee om Hospi al Clinic o Ba celona (HCB/2017/027). Pa ien s o a legal p oxy ga e w i en in o med consen p io o inclusion in he s udy. Clinical Assessmen Baseline demog aphic da a, pas medical his o y, clini- cal ea u es a SAH onse , and neu oimaging esul s we e p ospec i ely eco ded. Neu ological condi ion on admis- sion was assessed acco ding o he Wo ld Fede a ion o Neu osu gical Socie ies (WFNS) g ading sys em a e ini- ial esusci a ion. Pa ien s we e g ouped in o good g ade (WFNS g ades 1 and 2) o poo g ade (WFNS g ades 3 o 5) SAH. Pa ien s wi h SAH we e admi ed o he c i ical ca e uni (WFNS 3–5) o he S oke Uni (WFNS 1–2) as clinically app op ia e. Neu ological s a us [Glasgow Coma Scale (GCS) and Na ional Ins i u es Heal h S oke Scale (NIHSS)] we e assessed daily by neu ologis s in he S oke Uni o anes hesiologis s in he c i ical ca e uni . Sys emic and neu ological complica ions we e moni o ed and ea ed acco ding o in-hospi al p o ocols. GCS is a widely used assessmen ool o pa ien s’ le el o consciousness, based on h ee i ems: eye opening, e - bal esponse, and mo o esponse. I anges om 15 ( ull consciousness) o 3 (deep comma) [34]. WFNS is a SAH- speci ic g ading sys em ha classi ies pa ien s in o i e g oups, acco ding o hei GCS and p esence o absence o mo o de ici [35]. Thus, WFNS I ep esen s a SAH pa ien 1469Molecula Neu obiology (2025) 62:1467–1477 wi h unpai ed consciousness (GCS 15) and no mo o de ici , whe eas WFNS V ep esen s a coma ose pa ien (GCS 3 o 6). Las ly, NIHSS is a s anda dized assessmen ool mainly used in he con ex o ce eb o ascula diseases o e alua e ocal de ici s in se e al neu ological ields such as le el o consciousness, speech, mo o , and senso y unc ions. I anges om 0 (no de ici ) o 42 (maximum de ici ) and allows a p ecise and ep oducible moni o ing o he ex en o b ain damage [36]. All h ee scales ha e been widely epo ed in medical li e a u e in he con ex o SAH, wi h signi ican associa ions be ween wo se sco es and poo e p ognosis. Imaging Me hods Non-con as b ain CT was pe o med o all pa ien s a a i al. The ex en o suba achnoid bleeding was ca ego ized acco ding o he modi ied Fishe Scale (mF) classi ica ion. This scale ca ego izes he amoun o blood obse ed in non- con as b ain CT in o ou g oups, acco ding o bleeding hickness in suba achnoid space and he p esence o absence o in a en icula blood clo s. I anges om 1 ( hin suba- achnoid bleeding, no in a en icula blood) o 4 ( hick suba achnoid bleeding, in a en icula blood) and is posi- i ely co ela ed wi h he isk and se e i y o complica ions associa ed wi h he bleeding, mainly a e ial asospasm [37]. Addi ional ele an adiological ea u es including he p esence o in a en icula hemo hage, hyd ocephalus, o in apa enchymal hema oma we e also collec ed. Addi- ional imaging acquisi ions we e pe o med a disc e ion o he a ending physician acco ding o clinical e olu ion. An Angio-CT was pe o med o all pa ien s a a i al, as well as a ce eb al angiog aphy wi hin he i s 24h a e hospi al admission, as pa o ascula s udy. Acco ding o he p es- ence o absence o a b ain aneu ysm, he pa ien s we e u - he classi ied as non-aneu ysmal SAH o aneu ysmal SAH. In he la e g oup, da a on he numbe , loca ion, size, and ana omical ea u es o aneu ysms we e eco ded, and he index aneu ysm was e alua ed o endo ascula o su gical exclusion ea men acco ding o in-hospi al p o ocols. Sampling andLabo a o y Analysis Blood glucose was measu ed h ough inge p ick es ing a a i al and e e y 6h o a leas he ollowing 72h, as pa o usual clinical p ac ice. Glucose managemen consis ed on a sliding scale in as ing pa ien s and/o pa ien s ecei ing con inuous nu i ion h ough a nasogas ic ube and a basal- bolus insulin he apy in pa ien s ecei ing egula meals. Maximum, minimum, and mean glucose le els du ing he i s 72h we e eco ded. To e alua e se um NFL le els, an addi ional blood sam- ple (15mL) was d awn a 72h a e hospi al admission. The samples we e cen i uged a 4000 pm (1523 ela i e cen i ugal o ce) o 15min a 20–22°C, and supe na an was pipe ed and ozen a − 80°C un il u he analysis. NFL le els we e measu ed h ough he Simoa echnique (Quan e ix, Lexing on, MA, USA) in se um samples. In b ie , Simoa is a ou h-gene a ion ELISA based on p o eic an igen cap u ing by pa amagne ic mic oscopic beads ha ca y sandwich an ibody complexes. Then, hose beads a e p ecipi a ed in o single-molecule a ays ha a e indi idu- ally ead o check o hei bond o he index an igen. Ba ch se um NFL measu emen was pe o med acco ding o he manu ac u e ’s guide. Clinical Ou come E alua ion The p ede ined p ima y ou come measu e was unc ional disabili y, e alua ed wi h he modi ied Rankin Scale (mRS). Func ional ou come was assessed by ce i ied neu ologis s on in-pe son isi s a he ou pa ien clinic 90days a e ini ial hospi al admission, h ough a semi-s uc u ed clini- cal in e iew. Poo unc ional ou come was p ede ined as mRS > 2. S a is ical Analysis Con inuous a iables a e shown as mean (s anda d de ia- ion) o median (in e qua ile in e als), and di e ences be ween g oups we e assessed using S uden ’s - es , ANOVA, o Mann–Whi ney as app op ia e. Ca ego ical a - iables a e shown as absolu e alues (pe cen age) and com- pa ed using chi-squa ed o Fishe ’s exac es s. A uni a ia e analysis was un o assess he associa ion o demog aphic, clinical, and ea men - ela ed a iables wi h clinical ou - come a 3mon hs and wi h NFL le els. The accu acy o he explo ed glucose me ics o p edic ing clinical ou come was assessed wi h ecei e ope a ing cha ac e is ic cu es (ROC), and he glucose me ic wi h highe a ea unde he cu e (AUC) was selec ed o i s inclusion in mul i a ia e models. A mul i a ia e logis ic eg ession model analyzed he p edic o s o poo clinical ou come, and a mul i a ia e linea eg ession assessed he a iables associa ed wi h NFL le els. Spea man’s co ela ion analyses we e pe o med o assess he associa ions be ween glucose me ics, NFL le - els, and mRS sco es, and he nume ical esul s we e illus- a ed h ough a co ela ion ma ix. Fo linea eg ession and media ion analyses, we used log- ans o med NFL le els o app oach no mali y. Age and WFNS a admission we e o ced o emain in he inal model o hei p ognos ic el- e ance based on p io knowledge. To a oid model o e i - ing, a backwa d elimina ion me hod (likelihood a io) was used o selec he inal bina y logis ic eg ession model, and he Hosme –Lemeshow goodness-o - i s a is ic was used o assess he inal model i . Media ion analyses we e also 1470 Molecula Neu obiology (2025) 62:1467–1477 implemen ed o assess whe he glucose in luenced clinical ou come indi ec ly ia NFL by using he PROCESS mac o (Hayes, 2013) o he s a is ical so wa e IBM SPSS S a is- ics o Windows (IBM, A monk, NY, USA); 10,000 boo - s aps p oduced 95% con idence in e als (CI) on he media- ion coe icien pa ame e es ima es, and media ed (indi ec ) e ec s we e conside ed signi ican i he 95% CI o hei pa ame e es ima es did no c oss he ze o alue. The analy- ses we e pe o med using SPSS e sion 25.0, and he le el o signi icance was es ablished a a 0.05 le el ( wo-sided). Da a A ailabili y The da ase s gene a ed and analyzed a e a ailable om he co esponding au ho s on easonable eques . Resul s Cha ac e is ics o  heS udy Popula ion A o al o 92 pa ien s we e included du ing he s udy pe iod. The main ai s o he included pa ien s a e shown in Table1. O e all, 61 (66%) pa ien s we e women wi h a median (IQR) age o 55 (49–65) yea s. On admission, 36 (39%) pa ien s p esen ed wi h poo clinical g ade SAH (WFNS 4–5), and 67 (73%) had a modi ied Fishe Scale sco e o 4. A b ain aneu ysm was ound in 84 (91%) pa ien s, om whom 57 (62%) we e ea ed endo ascula ly and 23 (25%) ecei ed su gical clipping. Ini ial neu oimaging assessmen e ealed he p esence o hyd ocephalus in 58 (63%) pa ien s, pa en- chymal hemo hage in 22 (24%), and in a en icula bleed- ing in 68 (74%). Table 1 S udy popula ion cha ac e is ics assessed du ing he ea ly b ain inju y pe iod acco ding o clinical ou come a 3mon hs (whole sample) Quali a i e da a a e exp essed as n (%) and quan i a i e da a as median (IQR) mRS modi ied Rankin Scale, SD s anda d de ia ion mRS 0–2 (n = 60) mRS 3–6 (n = 32) p Demog aphics Age (yea s) 54 (49–62) 61 (52–74) 0.054 Female sex 39 (65) 22 (69) 0.717 P e-mo bid mRS 0 (0–0) 0 (0–1) 0.003 Medical his o y Hype ension 21 (35) 17 (53) 0.093 Dyslipidemia 9 (15) 8 (25) 0.239 Baseline ai s WFNS g ade (4–5) 13 (22) 23 (72) < 0.001 Aneu ysm (yes) 54 (90) 30 (94) 0.543 Aneu ysm size (mm) 5 (4–6) 6 (5–9) 0.010 Modi ied Fishe Scale 4 (3–4) 4 (4–4) 0.033 In a en icula bleeding 41 (68) 27 (84) 0.095 Hyd ocephalus 33 (55) 25 (68) 0.029 Pa enchymal hemo hage 13 (22) 9 (28) 0.489 Global ce eb al edema 33 (55) 20 (63) 0.488 Aneu ysm ea men modali y 0.596 No 8 (13) 4 (13) Endo ascula 39 (65) 18 (56) Su gical 13 (22) 10 (31) Glucose me ics Glucose a admission (mg/dL) 125 (113–142) 138 (126–164) 0.005 Mean glucose wi hin 72h (mg/dL) 114 (103–128) 137 (123–150) < 0.001 Max glucose wi hin 72h (mg/dL) 150 (126–164) 165 (150–188) 0.001 Min glucose wi hin 72h (mg/dL) 87 (81–100) 103 (97–121) < 0.001 SD glucose wi hin 72h (mg/dL) 18 (14–24) 23 (16–28) 0.106 1471Molecula Neu obiology (2025) 62:1467–1477 Glucose Me ics Du ing heEBI Pe iod Glucose me ics du ing he EBI pe iod including glucose a admission, maximum, minimum, mean, and s anda d de ia ion o glucose le els a e shown in Table1. In uni- a ia e analyses, all glucose me ics excep he s anda d de ia ion (SD) we e associa ed wi h clinical ou come a 90days. In bina y logis ic eg ession mul i a ia e analy- ses adjus ed by baseline clinical se e i y (WFNS), only mean glucose le els and minimum glucose le els wi hin he i s 72h a e bleeding onse emained associa ed wi h poo clinical ou come a 90days [mean glucose le els, OR (95% CI) pe each mg/dL o inc ease 1.04 (1.010–1.077), p = 0.010; minimum glucose le els, OR (95% CI) pe each mg/dL o inc ease 1.04 (1.007–1.079), p = 0.020]. In ag eemen wi h hese obse a ions, in ROC analysis, mean glucose le els du ing he EBI pe iod disclosed he high- es AUC alue, ollowed by minimum glucose du ing he EBI pe iod, maximum glucose du ing he EBI pe iod, and glucose le els a hospi al admission, as shown in Table2. Acco ding o hese indings, mean glucose le els du ing he i s 72h a e hospi al admission we e used o u he analyses. Va iables Associa ed wi hPoo Func ional Ou come In his coho , a o al o 32 (35%) pa ien s showed poo unc ional ou come a 90days. In uni a ia e analyses, poo ou come was associa ed wi h wo se p emo bid mRS, poo e ini ial WFNS g ade, la ge aneu ysm size, highe mF sco e, and hyd ocephalus a ini ial neu oimag- ing, as well as wi h highe glucose le els du ing he i s 72h (Table1). In mul i a ia e models, he a iables ha emained independen ly associa ed wi h poo clinical ou - come we e poo e WFNS g ade a onse and highe mean glucose le els du ing he EBI pe iod (Table3). Ci cula ing NFL Le els a  heEnd o  heEBI Pe iod: Con ibu o s andAssocia ion wi hClinical Ou come The median (IQR) le els o NFL a he end o he EBI pe iod we e 25 (13–56) pg/mL. The a iables associa ed wi h highe NFL le els in uni a ia e analysis included olde age, poo e ini ial WFNS g ade, and highe mean glucose le els du ing he EBI pe iod, as shown in Table4. In mul i a i- a e analyses, only glucose le els emained associa ed wi h NFL [adjus ed OR 1.01 (95% CI 1.001–1.024, p = 0.03)]. The le els o NFL we e signi ican ly highe in pa ien s wi h poo clinical ou come [median (IQR) 56 (26–126) e sus 19 (11–32) in pa ien s wi h poo and good clinical ou come, espec i ely; p < 0.001], as shown in Fig.1A. A ma ix o co ela ions be ween glucose me ics assessed du - ing he EBI pe iod, NFL le els measu ed a 72h, and mRS a 3mon hs is shown in Fig.1B. O no e, he associa ion be ween NFL le els and poo clinical ou come emained signi ican in bina y eg ession models adjus ed by age, p e-mo bid mRS, WFNS g ade a hospi al admission, and mean glucose le els du ing he EBI pe iod [adjus ed OR 2.61 (95% CI 1.364–5.010, p = 0.004)]. Glucose Le els Du ing heEBI Pe iod, Ci cula ing NFL Le els a  heEnd o  heEBI Pe iod, andClinical Ou come a 90 Days: AMedia ion Analysis A media ion analysis was implemen ed o e alua e whe he he associa ion be ween highe glucose le els du ing he EBI pe iod and clinical ou come was media ed h ough i s asso- cia ion wi h inc eased NFL le els, as schema ically shown in Fig.2A. As shown in Fig.2B, in unadjus ed analyses, glu- cose le els du ing EBI we e associa ed wi h highe ci cula - ing NFL le els a 72h (media o a iable), and bo h glucose and NFL le els we e also signi ican ly associa ed wi h poo clinical ou come. Media ion analysis be ween glucose le els Table 2 Recei e ope a ing cha ac e is ic cu e analyses o he accu- acy o glucose me ics o p edic ing clinical ou come a 90days AUC a ea unde he cu e, CI con idence in e al, SD s anda d de ia- ion Glucose me ics AUC (95% CI), p p alue Glucose a admission 0.680 (0.569–0.792) 0.005 Mean glucose wi hin 72h 0.797 (0.706–0.889) < 0.001 Max glucose wi hin 72h 0.708 (0.600–0.817) 0.001 Min glucose wi hin 72h 0.764 (0.664–0.863) < 0.001 SD glucose wi hin 72h 0.605 (0.481–0.729) 0.098 Table 3 Va iables assessed du ing EBI pe iod associa ed wi h poo clinical ou come (mRS > 2): mul i a ia e analysis The a iables ha emained in he inal model we e selec ed h ough bina y logis ic eg ession wi h backwa d p ocedu e (likelihood a io). The ini ial model included all he a iables showing a p alue o < 0.1 in uni a ia e analysis (shown in Table1). Age and WFNS a admis- sion we e o ced o emain in he inal model OR odds a io, mRS modi ied Rankin Scale, IQR in e qua ile ange, WFNS Wo ld Fede a ion o Neu osu gical Socie ies Scale Va iables OR (95% CI) p alue Age (pe yea ) 1.02 (0.980–1.069) 0.303 P e-mo bid mRS (pe IQR) 1.90 (0.932–3.865) 0.077 WFNS g ade (4–5) 4.05 (1.276–12.877) 0.018 Mean glucose wi hin 72h (pe mg/ dL) 1.04 (1.009–1.081) 0.014 1472 Molecula Neu obiology (2025) 62:1467–1477 and poo clinical ou come a 90days e ealed a signi ican indi ec e ec (coe icien = 0.02, 95% CI = 0.007–0.041, p < 0.005) indica ing ha he associa ion be ween glucose and clinical ou come was in pa media ed by NFL le els. A e adjus men o he media o NFL, glucose s ill had a subs an ial associa ion wi h unc ional ou come. O e all, in unadjus ed analyses, he media o NFL explained 29% o he associa ion o highe glucose wi h poo unc ional ou come. In analyses adjus ed by age, WFNS a hospi al admission and p e-mo bid mRS, he associa ions be ween glucose, Table 4 Uni a ia e and mul i a ia e analyses o he associa ions be ween se um NFL le els (log- ans o med) and baseline a iables OR odds a io, CI con idence in e al, mRS modi ied Rankin Scale, IQR in e qua ile ange, WFNS Wo ld Fede a ion o Neu osu gical Socie ies Scale Va iables Uni a ia e analysis OR (95% CI), p alue Mul i a ia e analysis OR (95% CI), p alue Age (pe yea ) 1.02 (1.000–1.034), p = 0.045 1.01 (0.996–1.027), p = 0.162 Female sex ( s male) 1.22 (0.765–1.928), p = 0.409 - P e-mo bid mRS (pe IQR) 1.17 (0.874–1.575), p = 0.288 - Hype ension (yes) 1.27 (0.814–1.972), p = 0.294 - Dyslipidemia (yes) 1.52 (0.867–2.646), p = 0.145 - WFNS g ade (4–5) 2.11 (1.385–3.222), p = 0.001 1.51 (0.926–2.463), p = 0.098 Aneu ysm (yes) 1.52 (0.701–3.287), p = 0.290 - Aneu ysm size (pe IQR) 1.13 (0.925–1.379), p = 0.233 - Modi ied Fishe Scale (pe IQR) 1.10 (0.862–1.399), p = 0.448 - In a en icula bleeding (yes) 1.09 (0.659–1.786), p = 0.750 - Hyd ocephalus (yes) 1.21 (0.770–1.902), p = 0.408 - Pa enchymal hemo hage (yes) 1.61 (0.970–2.661), p = 0.065 1.34 (0.834–2.151), p = 0.227 Global ce eb al edema (yes) 1.08 (0.696–1.688), p = 0.722 - Aneu ysm ea men modali y (pe ype) 1.03 (0.757–1.397), p = 0.858 - Mean glucose wi hin 72h (pe mg/dL) 1.02 (1.011–1.030), p < 0.001 1.01 (1.001–1.024), p = 0.039 Fig. 1 A NFL le els a 72h a e admission in pa ien s wi h good (mRS 0–2) and poo (mRS 3–6) ou come a 3mon hs. B Co ela ion ma ix be ween mean glucose in 72h, NFL le els a 72h, and poo clinical ou come a 3mon hs. Nume ical da a wi hin he ma ix ep- esen Spea man’s ank co ela ion coe icien s; single as e isk means p alue lowe han 0.05; double as e isks mean p alue lowe han 0.01. NFL, neu o ilamen ligh chain; mRS, modi ied Rankin Scale; SD, s anda d de ia ion 1473Molecula Neu obiology (2025) 62:1467–1477 NFL, and clinical ou come emained signi ican , as shown in Fig.2C. Adjus ed media ion analyses e ealed no signi ican di ec e ec o glucose on unc ional ou come, while he e was a signi ican indi ec e ec ha was media ed by NFL le els (coe icien = 0.01, 95% CI = 0.001–0.040, p < 0.005). O e all, in adjus ed analyses, he media o NFL explained 25% o he associa ion o highe glucose le els wi h poo unc ional ou come. Discussion In ag eemen wi h p e ious epo s, in his coho o SAH pa ien s, highe glucose le els du ing he EBI pe iod we e associa ed wi h poo clinical ou come a long e m. Acu e hype glycemia du ing he EBI pe iod also p edic ed highe ci cula ing NFL le els measu ed a he end o he EBI pe iod. Acco ding o media ion analyses, he associa ion be ween glucose and poo clinical ou come was signi ican ly media ed h ough he link be ween glucose and NFL le els. These obse a ions sugges ha he associa ion be ween hype glycemia and poo clinical ou come in SAH migh be explained in pa h ough glucose-d i en seconda y NAD. In line wi h cu en knowledge, in his coho , glucose le els measu ed du ing he EBI pe iod a e SAH onse eme ged as one o he mos obus p edic o s o poo ou - come, along wi h inc eased clinical se e i y a hospi al admission [15–20]. In ou sample, all he explo ed glu- cose me ics excep he s anda d de ia ion we e associ- a ed wi h clinical ou come a 90days. Acco ding o ROC analysis, mean glucose le els du ing he EBI pe iod disclosed he highes accu acy o p edic ing poo clini- cal ou come a 3mon hs, especially in compa ison wi h admission glucose le els. This obse a ion is in line wi h p e ious epo s. None heless, we ound no associa ion be ween s anda d de ia ion and p ognosis in ou coho , whe eas he e is inc easing e idence ha dynamic glucose pa ame e s du ing he i s days a e s oke could be mo e in o ma i e o he p edic ion o in hospi al complica ions o long- e m clinical ou come han isola ed ini ial meas- u emen s [16, 17, 38–42]. This could be explained due o he limi ed empo al esolu ion o glucose me ics de i ed om cu en s anda ds o ca e, hus limi ing he powe o assessmen s ega ding glucose dynamics and p ogno- sis. In his con ex , he use o comp ehensi e dynamic e alua ions such as hose de i ed om con inuous glu- cose moni o ing de ices could aid o ob ain a mo e p ecise Fig. 2 Model o he media ion pa hways be ween glucose le els wi hin he i s 72h a e bleeding onse (glucose du ing EBI) and clinical ou come a day 90 acco ding o ci cula ing NFL le els (log- ans o med) measu ed a 72h a e bleeding (NFL a 72h). A Theo- e ical amewo k o a pa hway model designed o explain he media- ion e ec o NFL on he associa ion be ween glucose and clinical ou come a day 90 (dicho omized mRS sco e; poo ou come de ined as mRS > 2). B, C Unadjus ed and adjus ed pa h media ion analyses, espec i ely. Coe icien s in he pa h models a e lis ed as a, b, and c′, whe e a and b a e pa o he indi ec pa h and c′ is he di ec pa h adjus ed o he indi ec pa h; he o al e ec is he composi ion o he di ec e ec (c′) and he indi ec e ec (ab). Da a a e coe icien es i- ma es wi h 95% con idence in e als. EBI, ea ly b ain inju y; NFL, neu o ilamen ligh chain; mRS, modi ied Rankin Scale; M, media o 1474 Molecula Neu obiology (2025) 62:1467–1477 knowledge on he p ognos ic and physiopa hological el- e ance o glycemic pa e ns du ing he acu e phase o SAH [42]. In his s udy, we e alua ed he p esence o NAD non- in asi ely h ough he measu emen o ci cula ing NFL le els a he end o he EBI pe iod. The e o e, ci cula ing NFL le els we e used as a su oga e ma ke o consequence o ongoing b ain inju y du ing he EBI pe iod and no as a d i e (o di ec cause) o poo clinical ou come a long e m. Up o less han a decade ago, NFL could only be eli- ably measu able in CSF due o echnical limi a ions. The de elopmen o he Simoa echnique allows NFL measu e- men in pe iphe al biological samples, hen making possible an in-dep h e alua ion o NFL as a bioma ke o disease se e i y and p ognosis. Acco ding o p e ious epo s, in he se ing o SAH, highe NFL le els co ela e consis en ly wi h wo se clinical and adiological se e i y o he bleed- ing a admission as well as wi h poo clinical ou come a long e m [25–33]. In ou coho , we con i med he associa- ion be ween se um NFL le els and poo clinical ou come a 3mon hs a e bleeding. As a no el inding, we ound ha highe se um NFL le els measu ed a he end o he EBI pe iod we e p edic ed by highe glucose le els meas- u ed du ing he acu e phase a e bleeding in adjus ed linea eg ession analysis, independen ly o age o clinical s a us a hospi al admission. Gi en he signi ican mul ila e al asso- cia ions ound be ween glucose le els du ing he EBI pe iod, NFL le els measu ed a he end o he EBI pe iod, and clini- cal ou come a 90days, we implemen ed a media ion analy- sis o explo e whe he he associa ion be ween glucose and poo clinical ou come was media ed h ough NFL le els as a media o a iable. Acco ding o hese analyses, he associa- ion o glucose wi h NFL le els was s onge han i s di ec associa ion wi h clinical ou come, hus sugges ing ha he e ec o glucose on long- e m clinical ou come was a leas in pa media ed by he magni ude o neu oaxonal inju y. Indeed, he indi ec media ion pa hway explained be ween 29 and 25% o he associa ion be ween glucose and clinical ou come a day 90 in unadjus ed and adjus ed media ion analyses, espec i ely. O e all, hese da a sugges ha ele- a ed glucose le els du ing he EBI pe iod migh hampe he clinical eco e y a long e m by p omo ing glucose-d i en neu oaxonal inju y. This suppo s he concep ualiza ion o glucose as an ac i e agen in neu onal damage a he han a passi e epiphenomenon o disease se e i y. Howe e , as media ion analyses do no imply causali y, he alidi y and di ec ion o hese ela ionships dese e u he in es iga ion in addi ional la ge p ospec i e obse a ional coho s udies and in in e en ional p eclinical and clinical ials. Acco d- ing o ou indings, he use o su oga e ma ke s o NAD such as NFL le els could be aluable o he assessmen o he biological e ec o in e en ional ials aimed o imp o e glycemic con ol o speci ic pha macological agen s. The main s eng h o his s udy was he use o a well-cha - ac e ized p ospec i e coho o consecu i e SAH subjec s admi ed in a Comp ehensi e S oke Cen e . Fu he mo e, NFL le els we e assessed h ough a well- alida ed ou h gene a ion Simoa echnique. Also, he media ion analysis app oach, e en i no causa ion is implied, sheds ligh on he po en ial glucose-d i en ha m ul pa hways igge ed du ing EBI pe iod. Ne e heless, his s udy has se e al limi a ions. Fi s , we used unadjus ed and adjus ed media ion analyses o cha ac e ize o he associa ion be ween hype glycemia, NAD, and clinical ou come in ou coho o SAH pa ien s. Gi en he obse a ional na u e o his s udy, he es ima ed di ec and indi ec e ec s o he associa ion be ween hype glycemia and clinical ou come may ha e been a ec ed by unmeasu ed con- ounding. The e o e, he media ion e ec ound in his s udy should be in e p e ed wi h cau ion and equi es u he con i - ma ion in ex e nal obse a ional coho s and in in e en ional s udies [43]. Second, NFL le els we e assessed a he end o he EBI pe iod as a su oga e bioma ke o b ain inju y o e lec he amoun o NAD occu ing du ing he whole EBI pe iod. Consequen ly, ou esul s migh be only applicable o he subse o SAH pa ien s who su i e beyond 72h a e bleeding onse . Thi d, his was an obse a ional s udy whe e hype glycemia was managed acco ding o local in-hospi al p o ocols and he e o e a cause-and-e ec ela ionship canno be in e ed om he ob ained da a. Fou h, NAD was exclu- si ely e alua ed using one single blood bioma ke in a single ime poin . The assessmen o longi udinal se um NFL le els and/o addi ional NAD su oga e bioma ke s such as hose de i ed om ad anced quan i a i e MRI including mic o as- cula , me abolic, and mic os uc u al in eg i y measu emen s could be highly in o ma i e o iden i ying glucose p o iles associa ed wi h highe b ain damage a e SAH. A be e knowledge on he link be ween ad anced quan i a i e neu o- imaging me ics o NFL le els and longi udinal glucose p o- iles du ing he EBI pe iod is needed o unde s and he mos app op ia e and in o ma i e iming and how o combine he di e en su oga e ma ke s o acu e b ain inju y. E en ually, his in o ma ion may lead o imp o e glucose managemen p o ocols and o op imize he design o clinical ials aimed o modula e glucose le els in he acu e phase a e SAH. Finally, his epo lacks addi ional mechanis ic s udies on molecula le els o explain how high glucose le els lead o inc eased NFL le els. Fu he p eclinical and clinical s udies a e needed o con i m his ela ionship and o unde s and he mechanis ic link be ween glucose dis u bances and neu oaxonal damage in his disease. 1475Molecula Neu obiology (2025) 62:1467–1477 Conclusions Bo h highe mean glucose measu ed along he EBI pe iod and highe NFL le els measu ed a he end o he EBI pe iod we e independen ly associa ed wi h poo clinical ou come a 90days. The media ion analyses sugges ed ha app oxi- ma ely 25% o he a iabili y o he associa ion be ween glucose and poo p ognosis could be media ed h ough neu- oaxonal damage as measu ed ia ci cula ing NFL le els. These esul s sugges ha he link be ween hype glycemia and poo clinical ou come may be pa ly explained by sec- onda y neu oaxonal inju y. Addi ional esea ch is needed o con i m hese associa ions and o explo e he mechanis ic link be ween hype glycemia and seconda y b ain damage. Supplemen a y In o ma ion The online e sion con ains supplemen- a y ma e ial a ailable a h ps:// doi. o g/ 10. 1007/ s12035- 024- 04347-6. Acknowledgemen s We hank he Spanish Minis y o Economy and Compe i i eness, Ins i u o de Salud Ca los III, and he Eu opean Regional De elopmen Fund (ERDF) o he inancial suppo o he co esponding au ho s. We also hank Fundació La Ma a ó de TV3 o he inancial suppo o Se gio Ama o. We hank Hospi al Clinic de Ba celona as well o he inancial suppo o he i s au ho . Au ho Con ibu ion Se gio Ama o, Ramón To né, and Ángel Cham- o o con ibu ed o he s udy concep ualiza ion. Daniel San ana, Ale- jand a Mos ei o, Gab iel Pujol, Lau a Llull, Ma iano We ne , and Luigi Za e a we e in cha ge o ma e ial p epa a ion and da a collec ion. Ab aham Ma ín and Ca les Jus icia ad ised abou neu o ilamen meas- u emen echniques. Da a analysis was pe o med by Se gio Ama o. Lei e Ped osa gene a ed he igu es o he manusc ip . The i s d a o he manusc ip was w i en by Daniel San ana, and all au ho s com- men ed on p e ious e sions o he manusc ip . All au ho s ead and app o ed he inal manusc ip . Funding Open Access unding p o ided hanks o he CRUE-CSIC ag eemen wi h Sp inge Na u e. Se gio Ama o and Ramón To né ecei ed inancial suppo om a g an gi en by he Spanish Minis- y o Economy and Compe i i eness [p ojec PI19/00936 unded by Ins i u o de Salud Ca los III and co- unded by he Eu opean Regional De elopmen Fund (ERDF)]. Se gio Ama o also ecei ed inancial suppo om a g an gi en by Fundació la Ma a ó de TV3 (g an num- be 17/C/2017). Daniel San ana is suppo ed by a g an om Hospi al Clinic de Ba celona (Con ac e Clínic de Rece ca Emili Le ang-Josep Fon ). Da a A ailabili y The da ase s gene a ed and analyzed a e a ailable om he co esponding au ho s on easonable eques . Decla a ions E hics App o al This s udy was pe o med in line wi h he p inciples o he Decla a ion o Helsinki. E hics app o al was ob ained om he local Clinical Resea ch E hics Commi ee om Hospi al Clinic o Ba - celona (HCB/2017/027). Consen o Pa icipa e Pa ien s o a legal p oxy ga e w i en in o med consen p io o inclusion in he s udy. Consen o Publica ion No applicable. No indi idual pe son’s da a in any o m is shown. Compe ing In e es s The au ho s decla e no compe ing in e es s. Open Access This a icle is licensed unde a C ea i e Commons A i- bu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a- ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. The images o o he hi d pa y ma e ial in his a icle a e included in he a icle’s C ea i e Commons licence, unless indica ed o he wise in a c edi line o he ma e ial. 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